NO318764B1 - Human <beta> <N> 2 </N> integrin alpha subunit - Google Patents

Description

The present patent application is a partial continuation

of US Patent Application No. 08/362,652, filed December 21, 1994, which in turn is a continuation-in-part of US Patent Application No. 08/286,889, filed August 5, 1994, issued as US Patent No. 5,470,953 on November 28, 1995, which in turn is a continuation in part of US Patent Application No. 08/173,497, filed December 23, 1993, issued as US Patent No. 5,437,958 on

1 August 1995.

Field of the invention

The present invention relates to the hybridoma cell line 199M with accession number ATCC HB-12058 and associated antibody for a dd-binding protein which is a new human S2-integrin-a subunit that is structurally related to the known human {32-integrin-a-subunits CDlla, CDllb and CDllc.

The background of the invention

The integrins are a class of membrane-associated molecules that actively participate in cellular adhesion. Integrins are transmembrane heterodimers comprising an α-subunit in non-covalent association with a β-subunit. To date, at least 14 α-subunits and 8 β-subunits have been identified [described by Springer, Nature, 346:425-434 (1990)]. The £}-"subunits are generally capable of association with more than one α-subunit,

and the heterodimers that share a common β-subunit have been classified as subfamilies within the integrin population.

One class of human integrins, restricted to expression in white blood cells, is characterized by a common β2 subunit. As a result of this cell-specific expression, these integrins are commonly referred to as leukocyte integrins, Leu-CAM or leukointegrins. Because of the common |32 subunit, an alternative term for this class is |32 integrins. The p2 subunit (CD18) has previously been isolated in connection with one of three distinct α subunits, CDlla, CDllb or CDllc. The isolation of a cDNA encoding human CD18,

is described by Kishimoto et al., Cell, 48:681-690 (1987). With official WHO nomenclature, the heterodimeric proteins are referred to

to as CDlla/CD18, CDllb/CD18 and CDllc/CD18; in common nomenclature they are referred to as LFA-1, Mac-1 or Mol and pl50,95 or LeuM5 respectively [Cobbold et al., in Leukocyte Typing III, McMichael (ed.), Oxford Press, p. 788 (1987)] . The human pvintegrin-α subunits CDlla, CDllb and CDllc have been shown to migrate under reducing conditions by electrophoresis with apparent molecular weights of approx. 180 kD, 155 kD and 150 kD, and DNA encoding these subunits have been cloned [CDlla, Larson et al., J. Cell Biol., 108:703-712

(1989); CD11b, Corbi et al., J. Biol. Chem., 263:12403-12411

(1988) and CDllc, Corbi et al., EMBO J., 6:4023-4028 (1987)]. Putative homologues of the human £J2 integrin α and β chains, defined by approximate similarity in molecular weight, have been variously identified in other species, including monkeys and other primates [Letvin et al., Blood, 61: 408-410 (1983)], mice [Sanches-Madrid et al., J. Exp. Med., 154:1517 (1981)] and dogs [Moore et al., Tissue Antigens, 36:211-220 (1990) ].

The absolute molecular weights of putative homologues from other species have been shown to vary considerably [see e.g. Danilenko et al., Tissue Antigens, 40:13-21 (1992)], and in the absence of sequence information, a definitive correlation between human integrin subunits and those identified in other species has not been possible. Variation in the number of members in a protein family has also been observed between different species. One can e.g. considering that more IgA isotypes have been isolated in rabbits than in humans [Burnett et al., EMBO J., 8:4041-4047 (1989) and Schneiderman et al., Proe. Nati. Acad. Pollock. (USA), 86:7561-7565 (1989)]. In humans, at least six variants of the metallothionein protein have also previously been identified [Karin and Richards, Nature, 295:797-802

(1982) and Varshney et al., Mol. Cell. Biol., 6:26-37 (1986)], while in mice there is only evidence of two such variants [Searle et al., Mol. Cell. Biol., 4:1221-1230 (1984)]. Occurrence of many different members of a protein family in one species therefore does not necessarily imply that corresponding family members exist in another species.

In the particular context of fJ2 integrins, it has been observed in dogs that the putative canine p2 counterpart of the human CD18 is capable of dimerization with as many as four potentially different α-subunits [Danilenko et al., aupra]. Antibodies raised by immunization of mice with dog splenocytes resulted in monoclonal antibodies as immunoprecipitated proteins tentatively designated as dog homologues of human CD18, CDlla, CDllb and CDllc, mainly based on similar, but not identical, molecular weights. Another anti-canine splenocyte antibody, Call.8H2, recognized and immunoprecipitated a fourth canine α-like subunit that is also capable of association with the £2 subunit but has a unique molecular weight and is restricted in expression to a subset of differentiated tissue macrophages.

Antibodies raised by immunization of hamsters with mouse dendritic cells resulted in two anti-integrin antibodies [Metlay et al., J. Exp. Med., 171:1753-1771 (1990)]. One antibody, 2E6, immunoprecipitated an essentially heterodimer with subunits of approximate molecular weights of 180 kD and 90 kD, in addition to minor bands in the molecular weight range of 150-160 kD. The second antibody, N418, precipitated another apparent heterodimer with subunits of approximate molecular weights of 150 kD and 90 kD. Based on cell adhesion blocking studies, it was hypothesized that antibody 2E6 recognized a mouse counterpart of human CD18. Although the molecular weight of the N418 antigen suggested recognition of a mouse homolog of human CD11c/CD18, further analysis indicated that the mouse antigen exhibited a tissue distribution pattern inconsistent with that observed for human CD11c/CD18.

The antigens recognized by the canine Call.8H2 antibody and the mouse N418 antibody could represent a variant species (eg, a glycosylation or splice variant of a previously identified canine or mouse α-subunit. Alternatively, these antigens could represent unique canine - and mouse integrin α subunits In the absence of specific information regarding primary structure, these alternatives cannot be distinguished from each other.

In humans, CDlla/CD18 is expressed on all leukocytes. CDllb/CD18 and CDllc/CD18 are essentially restricted to expression on monocytes, granulocytes, macrophages and natural killer (NK) cells, but CDllc/CD18 is also detected on some B cell types. In general, CDlla/CD18 predominates on lymphocytes, CDllb/CD18 on granulocytes and CDllc/CD18 on macrophages [see review, Arnaout, Blood, 75:1037-1050 (1990)]. Expression of the α-chains is, however, variable with respect to the activation state and the differentiation of the individual cell types [see overview, Larson and Springer, Immunol. Rev., 114:181-217 (1990)].

The involvement of the β2 integrins in human immune and inflammatory responses has been demonstrated using monoclonal antibodies capable of blocking β2 integrin-associated cell adhesion. CDlla/CDl8, CDllb/CD18 and CDllc/CD18 participate e.g. active in natural killer (NK) cell binding to lymphomas and adenocarcinoma cells [Patarroyo et al., Immunol. Rev., 114:67-108 (1990)], granulocyte accumulation [Nourshargh et al., J. Immunol., 142:3193-3198 (1989)], granulocyte-independent plasma leakage [Arfors et al., Blood, 65:338 -340 (1987), chemotactic response of stimulated leukocytes [Arfors et al., aupra] and leukocyte adhesion to vascular endothelium [Price et al., J. Immunol., 139:4174-4177 (1987), and Smith et al., J. Clin. Invest., 83:2008-2017

(1989)]. The fundamental role of jp2 integrins in immune and inflammatory responses is made visible by the clinical syndrome referred to as leukocyte adhesion deficiency (LAD), in which clinical manifestations include recurrent and often life-threatening bacterial infections. LAD results from heterogeneous mutations in the β2 subunit [Kishimoto et al., Cell, 50:193-202 (1987)], and the severity of the disease state is proportional to the degree of failure in expression of the β2 subunit. Formation of the complete integrin heterodimer is impaired by the p"2 mutation [Kishimoto et al., supra].

Interestingly, at least one antibody specific for CD18 has been shown to inhibit human immunodeficiency virus type 1 (HIV-1) syncytium formation in vitro, although the exact mechanism for this inhibition is unclear [Hildreth and Orentas, Science, 244 :1075-1078 (1989)]. This observation is consistent with the discovery that a major counterreceptor for CDlla/CD18, ICAM-1, is also a surface receptor for the major group of rhinovirus serotypes [Greve et al., Cell, 56:839

(1989)].

In addition to the above-described role of β2-integrin, recently published data presented in WO 95/17412 describes the isolation of a new β2-integrin-α subunit also called Od-

The importance of β2 integrin binding activity in human immune and inflammatory responses thus understates the necessity to develop a more complete understanding of this class of surface proteins. Identification of hitherto unknown members of this integrin subfamily, as well as their counter-receptors, and the generation of monoclonal antibodies or other soluble factors that can alter biological activity of the β2 integrins, will provide practical means for therapeutic intervention in J32 integrin-related immune responses and inf lammatory responses.

Brief description of the invention

Hybridoma cell lines that produce antibodies specific for åa are covered by the present invention. Techniques for producing hybridomas secreting monoclonal antibodies are well known in the art. Hybridoma cell lines can be formed after immunization of an animal with purified dd, variants of Oa or cells expressing eid or a variant thereof on the extracellular membrane surface. Immunogenic cell types include cells that express Od in vivo, or transfected prokaryotic or eukaryotic cell lines that do not normally express dd in vivo. Currently preferred antibodies according to the invention are secreted by hybridomas with the designations 169A, 169B, 170D, 170F, 170E 170X, 170H, 188A, 188B, 188C, 188E, 188F, 188G, 1881, 188J, 188K, 188L, 18N, 188P, 188M, 188R, 188T, 195A, 195C, 195D, 195E, 195H, 197A-1, 197A-2, 197A-3, 197A-4, 199A, 199H AND 199M.

The present invention also relates to polypeptides and other non-peptide molecules that specifically bind to 0.3. Preferred binding molecules include antibodies (e.g., monoclonal and polyclonal antibodies), counterreceptors (e.g., membrane-associated and soluble forms), and other ligands (e.g., naturally occurring or synthetic molecules), including those molecules that competitively bind a<j i presence of ad-monoclonal antibodies and/or specific counter-receptors. Binding molecules are useful for purification of aa polypeptides and identification of cell types expressing Od. Binding molecules are also useful for modulating (ie inhibiting, blocking or stimulating) in vivo binding and/or signal transduction activities of da.

Novel purified and isolated polynucleotides (eg DNA and RNA transcripts, both sense and antisense strands) encoding a novel human β2 integrin α subunit, ad, and variants thereof (ie deletion, addition or substitution analogs) that possess binding and/or immunological properties inextricably linked to eid are described. Preferred DNA molecules include cDNA, genomic DNA, and wholly or partially chemically synthesized DNA molecules. A currently preferred polynucleotide is the DNA shown in SEQ ID NO. NO. 1, which codes for the polypeptide in SEQ ID NO. NO. 2. Recombinant plasmid and viral DNA constructs (expression constructs) that include Od coding sequences, wherein the id coding sequence is operably linked to a homologous or heterologous transcriptional regulatory element or elements, are also described.

Isolated and purified mouse and rat polynucleotides exhibiting homology to polynucleotides encoding human Od are also described. A preferred mouse polynucleotide is shown in SEQ ID NO. NO. 52; a preferred rat polynucleotide is shown in SEQ ID NO. NO. 54.

Prokaryotic or eukaryotic host cells transformed or transfected with DNA sequences expressing Od polypeptide or variants thereof are described. Host cells described are particularly useful for large-scale production of Od polypeptide which can be isolated either from the host cell itself or from the medium in which the host cell is grown. Host cells expressing Od polypeptide on their extracellular membrane surface are also useful as immunogens in the production of aa-specific antibodies. Host cells transfected with Od will preferentially be co-transfected to express a β2 integrin subunit to allow surface expression of the heterodimer.

Purified and isolated cia polypeptides, fragments and variations thereof are also described. Preferred ad polypeptides are as shown in SEQ ID NO. NO. 2. New aa products can be obtained as isolates from natural sources, but are preferably produced, together with da variant products, by recombinant procedures involving host cells. Fully glycosylated, partially glycosylated and fully deglycosylated forms of the aa polypeptide can be generated by varying the host cell selected for recombinant production and/or processing after isolation. Variant Oa polypeptides may include water-soluble and insoluble aa polypeptides, including analogs in which one or more of the amino acids have been deleted or replaced: (1) without loss, and preferably with gain, of one or more biological activities or immunological characteristics that are specific for Oa; or (2) with specific impairment of a particular ligand/receptor binding or signaling function. Fusion polypeptides are also described in which the ad amino acid sequences are expressed in conjunction with amino acid sequences from other polypeptides. Such fusion polypeptides may possess modified biological, biochemical and/or immunological properties compared to wild-type aa. Analogous polypeptides, including additional amino acid residues (e.g., lysine or cysteine) that facilitate multimer-

formation, is included.

Assays to identify Oa binding molecules are also described, including immobilized ligand binding assays, solution binding assays, late-tilt proximity assays, dihybrid screening assays, and the like.

In vitro assays for the identification of antibodies or other compounds that modulate the activity of cia, can e.g. involving immobilization of cia or a natural ligand to which oa binds, detectable labeling of the non-immobilized binding partner, incubation of the binding partners together, and determination of the effect of a test compound on the amount of bound label, wherein a reduction of label bound in the presence of the test compound compared with the amount of marker bound in the absence of the test compound indicates that the test compound is

an inhibitor of OA binding.

Another type of analysis for the identification of compounds that modulate the interaction between a<j and a ligand involves immobilizing eta or a fragment thereof on a solid support coated (or impregnated) with a fluorescent agent, labeling the ligand with a compound that is in capable of exciting the fluorescent agent, providing contact between the immobilized Od and the labeled ligand in the presence and absence of a putative modulator compound, detection of light emission from the fluorescent agent, and identification of modulatory compounds such as those compounds which affects the emission of light from the fluorescent agent, compared to the emission of light from the fluorescent agent in the absence of a modulating compound. Alternatively, the ad ligand can be immobilized, and the ad can be labeled in the assay.

A further method for the identification of compounds that modulate the interaction between dd and a ligand involves the transformation or transfection of suitable host cells with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor with a DNA binding domain and an activation domain, expression in the host cells of a first hybrid DNA sequence encoding a first fusion of part or all of Od and either the DNA binding domain or the activating domain of the transcription factor, expression in the host cells of a second hybrid DNA sequence which encodes part or all of the ligand and the DNA binding domain or the activation domain of the transcription factor not incorporated in the first fusion, evaluating the effect of a putative modulatory compound on the interaction between dd and the ligand, by detecting binding of the ligand to Od in a particular host cell by measuring the production of reporter gene product et in the host cell in the presence or absence of the putative modulator, and identification of modulatory compounds as those compounds that alter production of the reporter gene product compared to production of the reporter product in the absence of the modulatory compound. Particularly preferred for use in the assay are the 2 exA promoter, the lexA DNA binding domain, the Gal4 transactivation domain, the lacZ reporter gene and a yeast host cell.

A modified version of the above-described assay can be used for the isolation of a polynucleotide encoding a protein that binds to eid, by transformation or transfection of suitable host cells with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor with a DNA binding domain and an activation domain, expression in the host cells of a first hybrid DNA sequence encoding the first fusion of a part of, or all of, owned and either the DNA binding domain or the activation domain of the transcription factor, expression in the host cells of a library of other hybrid DNA sequences encoding other fusions of some, or all, putative Od binding proteins and the DNA binding domain or activation domain of the transcription factor not incorporated in the first fusion, detection of binding of an Ad binding protein to ad in a particular host cell upon detection of the production of the reporter gene product in the host cell n, and isolating other hybrid DNA sequences encoding Ad-binding protein from the particular host cell.

The value of the information provided by describing DNA and amino acid sequences for is self-evident. In a series of examples, the described Od cDNA sequence enables the isolation of the human aa genomic sequence, including the transcriptional control elements of the genomic sequence. Identification of Od allelic variants and heterologous DNA types (eg from rat or mouse) is also described. Isolation of the human Ad-genomic DNA and heterologous DNA types can be achieved by standard DNA/DNA hybridization techniques under suitable stringent conditions, using all or part of the Od cDNA sequence as a probe to screen a suitable library. Alternatively, polymerase chain reaction (PCR) using oligonucleotide primers designed based on the known cDNA sequence can be used to amplify and identify genomic ad-DNA sequences. Synthetic DNA encoding the cid polypeptide, including fragments and other variants thereof, can be produced by conventional synthetic methods.

DNA sequence information provided also enables development by homologous recombination or "knockout" strategies [see e.g. Kapecchi, Science, 244:1288-1292 (1989)] to produce rodents that do not express a functional Od polypeptide or that express a variant Od polypeptide. Such rodents are useful as models for studying the activities of cia and da modulators in vivo.

The DNA and amino acid sequences presented here also enable the analysis of Od epitopes which actively participate in counter-receptor binding, as well as epitopes which can regulate, instead of actively participating in, binding. Identification of epitopes that can participate in transmembrane signal transduction is also described.

The DNA described herein is also useful for detecting cell types that express ad polypeptide. Standard DNA/RNA hybridization techniques using Od DNA to detect Od RNA can be used to determine the basal level of Ad transcription in a cell, as well as changes in the level of transcription in response to internal or external agents. Identification of agents that modify transcription and/or translation of ad can in turn be assessed for potential therapeutic or prophylactic value. The DNA described here also enables in situ hybridization of Od DNA to cellular RNA to determine the cellular localization of oa-specific messages in complex cell populations and tissues.

The DNA described herein is also useful for the identification of non-human polynucleotide sequences that exhibit homology to human Od sequences. Possession of non-human Od DNA sequences allows the development of animal models (including, e.g., transgenic models) of the human system.

As another aspect of the invention, monoclonal or polyclonal antibodies specific for Ad can be used in immunohistochemical analysis to localize Od to subcellular compartments or individual cells in tissue. Immunohistochemical analyzes of this type are particularly useful when used in combination with in situ hybridization to locate both cid mRNA and polypeptide products of the ad gene. Identification of cell types that express oa can have significant ramifications for the development of therapeutic and prophylactic agents. It is expected that the products according to the invention related to eid can be used in the treatment of diseases in which macrophages are an essential element of the disease process. Animal models for many pathological conditions associated with macrophage activity are described in the art. In mice, e.g. macrophage recruitment present with both chronic and acute inflammation, reported by Jutila et al., J. Leukocyte Biol., 54:30-39 (1993). In rats, Adams et al.

[Transplantation, 53:1115-1119 (1992) and Transplantation, 56:794-799 (1993)] a model of transplant arteriosclerosis after heterotrophic abdominal heart allotransplantation. Rosenfeld et al. [Arteriosclerosis, 7:9-23 (1987) and Arteriosclerosis, 7:24-34 (1987)] describe induced atherosclerosis in rabbits fed a cholesterol-supplemented diet. Hanenberg et al. [Diabetologia, 32:126-134 (1989)] reports the spontaneous development of insulin-dependent diabetes in BB rats. Yamada et al. [Gastroenterology, 104:759-771 (1993)] describes an induced inflammatory bowel disease, chronic granulomatous colitis, in rats after injections of peptidoglycan-polysaccharide polymers from streptococci. Cromartie et al. [J. Exp. Med., 146:1585-1602 (1977)] and Schwab et al. [Infection and Immunity, 59:4436-4442 (1991)] reports that injection of cell wall protein from streptococci in rats leads to an arthritic condition characterized by inflammation of peripheral joints and subsequent joint destruction. Finally, Huitinga et al describe [Eur. J. Immunol., 23:709-715 (1993)] experimental allergic encephalomyelitis, a model of multiple sclerosis, in Lewis rats. In each of these models, dd antibodies, other Od-binding proteins or soluble forms of cia are used to weaken the disease state, presumably through inactivation of macrophage activity.

Pharmaceutical preparations for the treatment of these and other disease states are discussed here. Pharmaceutical preparations are designed for the purpose of inhibiting interaction between eid and its ligand(s), and include various soluble and membrane-associated forms of Oa (comprising the entire Qd polypeptide or fragments thereof that actively participate in eid binding), soluble and membrane-associated forms of oa binding proteins {including antibodies, ligands and the like), intracellular or extracellular modulators of Od binding activity and/or modulators of ad and/or ad ligand polypeptide expression, including modulators of transcription, translation, post-translational processing and/or intracellular transport.

Methods of treating disease states in which citi binding or localized accumulation of cells expressing eta is implicated in which a patient suffering from the disease state is administered an amount of a pharmaceutical composition sufficient to modulate levels of eta binding, or to modulate accumulation of cell types expressing cia is also discussed. The treatment method can be applied to disease states such as, but not limited to, type I diabetes, atherosclerosis, multiple sclerosis, asthma, psoriasis, lung inflammation, acute respiratory distress syndrome and rheumatoid arthritis.

Brief description of the figures

Countless other aspects and advantages of the present invention will become apparent when considering the following description with reference to the figures, in which: figures 1A-1D comprise an alignment of the human amino acid sequences of CDllb (SEQ. ID. NO. 3), CDllc (SEQ. ID. NO. 4) and then (SEQ. ID. NO. 2).

Detailed description of the invention

The present invention is illustrated by means of the following examples which relate to the isolation of a cDNA clone which codes for da from a human spleen cDNA library. Specifically, Example 1 illustrates the use of anti-dog ami antibody in an attempt to detect a homologous human protein. Example 2 describes in detail purification of canine Omi and N-terminal sequencing of the polypeptide to design oligonucleotide primers for PCR amplification of the canine α-mi gene. Example 3 describes large-scale purification of canine Omi for in-house sequencing to design additional PCR primers. Example 4 describes the use of PCR and the internal sequence primers to amplify a fragment of the canine Otmi gene. Example 5 describes the cloning of the human Od coding cDNA sequence. Example 6 describes Northern blot hybridization analyzes of human tissues and cells for expression of Od mRNA. Example 7 describes in detail construction of human Od expression plasmids and transfection of COS cells with the resulting plasmids. Example 8 describes ELISA analysis of Od expression in transfected COS cells. Example 9 describes FACS analysis of COS cells transfected with human" aa expression plasmids. Example 10 describes immunoprecipitation of CD18 in conjunction with ad in cotransfected COS cells. Example 11 relates to stable transfection of cid expression constructs in Chinese hamster ovary cells. Example 12 describes CD18-dependent binding of Od to the intercellular adhesion molecule ICAM-R, an ICAM-R mutant protein, and complement factor iC3b.Example 13 describes scintillation proximity screening assays to identify inhibitors or enhancers (ie, modulators) of dd ligand/antiligand binding interactions Example 14 describes the construction of expression plasmids encoding soluble forms of Od, and binding analyzes of the expression products. Example 15 relates to the production of Ad-specific, polyclonal sera and monoclonal antibodies. Example 16 describes analysis of Od tissue distribution, expression of Od on peripheral blood leukocytes and dd expression in inflammatory and non-inflammatory synovial fluid using anti-dd polyclonal serum. Example 17 describes the isolation of rat cDNA sequences showing homology to human dd gene sequences. Example 18 relates to construction of unabridged rat dd expression plasmids, rat od-1 domain expression plasmids, including I domain/IgG fusion proteins, and production of monoclonal antibodies against unabridged and I domain fusion proteins. Example 19 describes the isolation of mouse cDNA sequences showing homology to human dd gene sequences. Example 20 describes the isolation of additional mouse dd cDNA clones used to confirm the sequence analysis. Example 21 relates to in situ hybridization analysis of various mouse tissues to determine tissue- and cell-specific expression of the putative mouse homolog of human Oa. Example 22 describes generation of expression constructs encoding the putative mouse homologue of human EID. Example 23 describes the design of a "knockout" mouse in which the gene encoding the putative mouse homologue of human ad has been destroyed. Example 24 describes the isolation of rabbit cDNA clones showing homology to human Qd coding sequences. Example 25 describes animal models of human disease states in which modulation of dd is analyzed for therapeutic possibilities. Example 26 describes expression of Od in animal model disease conditions.

Example 1

Attempts to detect a human homologue of canine Otmi

The monoclonal antibody Call.8H2 [Moore et al.,

supra] , which is specific for canine a™., was tested for cross-reactivity on human peripheral blood leukocytes in an attempt to identify a human homologue of canine Orm- Cell preparations {typically 1 x 10<6> cells) were incubated with undiluted hybridoma supernatant or a purified mouse IgG negative control antibody (10 µg/ml) on ice in the presence of 0.1% sodium azide. Monoclonal antibody binding was detected by subsequent incubation with FlTC-conjugated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA) at 6 µg/ml. Stained cells were fixed with 2% w/v paraformaldehyde in phosphate-buffered saline (PBS) and analyzed with a Facstar Plus fluorescence-activated cell sorter (Becton Dickinson, Mountain View, CA). 10,000 cells were typically analyzed using logarithmic amplification for fluorescence intensity.

The results indicated that Call.8H2 did not cross-react with surface proteins expressed on human peripheral blood leukocytes, while the control cells, neoplastic canine peripheral blood lymphocytes, were essentially all positive for aTHi-

Because the monoclonal antibody Call.8H2 specific for the canine a-subunit did not cross-react with a human homolog, isolation of canine aTm DNA was considered a necessary prerequisite for isolating the human gene counterpart, if one existed.

Example 2

Affinity purification of canine aTHi for N-terminal sequencing

Hunde-Orm was affinity purified to determine N-terminal amino acid sequences for oligonucleotide probe/primer design. Briefly, anti-Orm monoclonal antibody Call.8H2 was coupled to Affigel 10 chromatographic resin (BioRad, Hercules, CA), and protein was isolated by specific antibody-protein interaction. Antibody was conjugated to the resin in accordance with the proposed protocol from BioRad, at a concentration of approx. 5 mg of antibody per ml of resin. After the conjugation reaction, excess antibody was removed and the resin was blocked with 3 volumes of 0.1 M ethanolamine. The resin was then washed with 30 column volumes of phosphate buffered saline (PBS). 25 g of a single dog spleen was homogenized in 250 ml of buffer containing 0.32 M sucrose in 25 mM Tris-HCl, pH 8.0, with protease inhibitors. Nuclei and degraded cell material were pelleted by centrifugation at 1000 g for 15 minutes. Membranes were pelleted from the supernatant by centrifugation at 100,000 g for 30 min. The membrane pellet was resuspended in 1,200 ml of lysis buffer (50 mM NaCl, 50 mM borate, pH 8.0, with 2% NP-40) and incubated for 1 h on ice. Insoluble material was then pelleted by centrifugation at 100,000 for 60 min. 10 ml of the clarified lysate was transferred to a 15 ml polypropylene tube with 0.5 ml of Cali.8H2-conjugated Affigel 10 resin, described above. The tube was incubated overnight at 4°C with rotation, and the resin was then washed with 50 column volumes of D-PBS. The resin was then transferred to a microcentrifuge tube and boiled for 10 min in 1 ml of Laemmli (non-reducing) sample buffer containing 0.1 M Tris-HCl, pH 6.8, 2% SDS, 20% glycerol, and 0.002% bromophenol blue. The resin was pelleted by centrifugation and discarded; the supernatant was treated with 1/15 volume of {3-mercaptoethanol (Sigma, St. Louis, MO) and chromatographed on a 7% polyacrylamide gel. The separated proteins were transferred to Immobilon PVDF membrane (Millipore, Bedford, MA) as follows.

The gels were washed once in deionized, Millipore-filtered water and equilibrated for 15–45 min in 10 mM 3-

[cyclohexylamino]-1-propanesulfonic acid (CAPS) transfer buffer, pH 10.5, with 10% methanol. Immobilon membranes were wetted with methanol, filled with filtered water and equilibrated for 15-30 min in CAPS transfer buffer. The initial transfer was performed using a Biorad transfer apparatus at 70 volts for 3 hours. The Immobilon membrane was removed after the transfer and stained in filtered 0.1% R250 Coomassie stain for 10 minutes. Membranes were destained in 50% methanol/10% acetic acid three times, 10 min each time. After decolorization was the membranes were washed in filtered water and air dried.

Protein bands of approx. 150 kD, 95 kD, 50 kD and 30 kD were detected. Presumably the 50 kD and 30 kD bands were the result of antibody contamination. N-terminal sequencing was then attempted on both the 150 kD and 95 kD bands, but the 95 kD protein was blocked, preventing sequencing. The 150 kD protein band was excised from the membrane and directly sequenced with an Applied Biosystems model 473A protein sequencer (Foster City, CA) according to the manufacturer's instructions. The resulting amino acid sequence is shown in SEQ ID NO.

NO. 5 using single letter amino acid designations.

The identified sequence included the FNLD sequence characteristic of α-subunits of the integrin family [Tamura et al., J. Cell. Biol., 111:1593-1604 (1990)].

Primer design and attempts to amplify canine ami sequences

From the N-terminal sequence information, three oligonucleotide probes were designed for hybridization: a) "Tommer", a fully degenerate oligonucleotide; b) "Patmer", a partially degenerate oligonucleotide; and c) "Guessmer", a non-degenerate oligonucleotide based on mammalian codon utilization. These probes are shown below as SEQ ID NOs, respectively. NO. 6, 7 and 8. Nucleic acid symbols are in accordance with 37 CF.R. §1.882 for these and all other nucleotide sequences herein.

Based on sequencing data, no relevant clones were detected using these oligonucleotides in multiple low-stringency hybridizations to a canine spleen/peripheral blood macrophage cDNA library cloned in AZAP (Stratagene, La Jolla,

ABOUT) .

Four other oligonucleotide primers, designated 5'Deg, 5'Spee, 3'Deg and 3'Spee (as shown in SEQ ID NO.

NO. 9, 10, 11 and 12, where Deg indicates degenerate and Spee indicates non-degenerate), were then designed based on the deduced N-terminal sequence to attempt to amplify canine aTHi sequences by PCR from phage library DNA purified from plate lysates of the Stratagene library described above. The ciTMi oligonucleotide primers were paired with the T3 or T7 vector primers, as shown in SEQ ID NO:2, respectively. NO. 13 and 14, which hybridize to sequences flanking the polylinker region of the Bluescript phagemid found in AZAP.

The PCR amplification was performed in Tag buffer (Boehringer Mannheim, Indianapolis, IN) containing magnesium, with 150 ng of library DNA, 1 µg of each primer, 200 µM dNTPs, and 2.5 units of Tag polymerase (Boehringer Mannheim), and the products were separated by electrophoresis on a 1% agarose gel in Tris-acetate-EDTA(TAE) buffer with 0.25 µg/ml ethidium bromide. DNA was transferred to a Hybond membrane (Amersham, Arlington Heights, IL) by wicking overnight in 10 x SSPE. After transfer, the immobilized DNA was denatured with 0.5 M NaOH with 0.6 M NaCl, neutralized with 1.0 M Tris-HCl, pH 8.0, in 1.5 M NaCl and washed with 2 x SSPE before UV cross-linking with a Stratalinker (Stratagene) cross-linking apparatus. The membrane was incubated in prehybridization buffer (5 x SSPE, 4 x Denhardt's, 0.8% SDS, 30% formamide) for 2 h at 50 °C with agitation.

Oligonucleotide probes 5'Deg, 5'Spec, 3'Deg and 3'Spee (SEQ ID NO: 9, 10, 11 and 12, respectively) were labeled using a Boehringer Mannheim kinase buffer with 100-300 uCi Y<p32 ->clATP and 1-3 units of polynucleotide kinase for 1-3 hours at 37 °C. Unincorporated label was removed by Sephadex G-25 fine chromatography (Pharmacia, Piscataway, NJ) using 10 mM Tris-HCl, pH 8.0, 1 mM EDTA(TE) buffer, and the eluate was added directly to prehybridization the solution. Membranes were probed for 16 h at 42 °C with agitation and washed repeatedly with a final stringency wash with 1 x SSPE/0.1% SDS at 50 °C for 15 min. The blot was then exposed with Kodak X-Omat AR film for 1-4 hours at -80°C.

The oligonucleotides 5'Deg, 5'Spee, 3'Deg and 3'Spee hybridized only to PCR products from the reactions in which they were used as primers, and did not hybridize as expected to PCR products from the reactions in which they were not used as primers . It was thus concluded that none of the PCR products were specific for aTMi because no product hybridized with all the probes.

Example 3

Large-scale affinity purification of canine ami for in-house sequencing

To provide an additional amino acid sequence for primer design, dog Orm was purified for internal sequencing. Three portions of frozen spleen (approximately 50 g each) and frozen cells from two dissected adult dog spleens were used to generate protein for internal sequencing. 50 g of spleen was homogenized in 200-300 ml of borate buffer with a Waring mixer. The homogenized material was diluted with 1 volume of buffer containing 4% NP-40, and the mixture was then gently stirred for at least 1 hour. The resulting lysate was cleared of large degradation fragments by centrifugation at 2000 g for 20 min, and then either filtered through a Corning prefilter (Corning, NY) or a Corning 0.8 micron filter. The lysate was further clarified by filtration through the Corning 0.4 micron filter system.

The spleen lysate and the antibody-conjugated Affigel 10 resin described in Example 2 were combined at a volume ratio of 150:1 in 100 ml aliquots and incubated overnight at 4°C on a rocker tray. The lysate was removed after centrifugation at 1000 g for 5 minutes, combined with more antibody-conjugated Affigel 10 resin and incubated overnight, as described above. The absorbed resin aliquots were then combined and washed with 50 volumes of D-PBS/0.1% Tween 20, and the resin was transferred to a 50 ml Biorad column. Adsorbed protein was eluted from the resin with 3-5 volumes of 0.1 M glycine (pH 2.5); fractions of approx. 900 µl was collected and neutralized with 100 µl 1 M Tris buffer, pH 8.0. Aliquots of 15 µl were removed from each fraction and boiled in an equal volume of 2x Laemmli sample buffer with 1/15 volume of 1 M dithiothreitol (DTT). These samples were electrophoresed on 8% Novex (San Diego, CA) polyacrylamide gels and visualized either by Coomassie staining or by silver staining using a Daiichi kit (Enprotech, Natick, MA) in accordance with the manufacturer's suggested protocol. Fractions containing the largest amounts of protein were combined and concentrated in vacuo. The remaining solution was diluted 50% with reducing Laemmli sample buffer and chromatographed on 1.5 mm 7% polyacrylamide gels in Tris-glycine/SDS buffer. Protein was transferred from the gels to Immobilon membrane by the procedure described in Example 2 using the Hoefer transfer apparatus.

The protein bands corresponding to dog-a™./ were excised from 10 PVDF membranes and gave approx. 47 ug total protein. The strips were destained in 4 ml of 50% methanol for 5 minutes, air dried and cut into 1 x 2 mm pieces. The membrane pieces were immersed in 2 mL of 95% acetone at 4 °C for 30 min with occasional vortexing

and then air dried.

Before proteolytic cleavage of the membrane-bound protein, 3 mg of cyanogen bromide (CNBr) (Pierce, Rockford, IL) was dissolved in 1.25 ml of 70% formic acid. This solution was then added to a tube containing the PVDF membrane pieces and the tube was incubated in the dark at room temperature for 24 hours. The supernatant (S1) was then transferred to another tube and the membrane pieces were washed with 0.25 ml of 70% formic acid. This supernatant (S2) was removed and added to the first supernatant (S1). 2 ml Milli Q water was added to the combined supernatants (S1 and S2) and the solution was lyophilized. The PVDF membrane pieces were dried under nitrogen and re-extracted with 1.25 mL of 60% acetonitrile, 0.1% tetrafluoroacetic acid (TFA) at 42 °C for 17 h. This supernatant (S3) was removed and the membrane pieces were re-extracted with 1.0 ml of 80% acetonitrile with 0.08% TFA at 42 °C for 1 h. This supernatant (S4) was combined with the previous supernatants (S1, S2 and S3) and vacuum dried.

The dried CNBr fragments were then dissolved in

63 µl 8 M urea, 0.4 M NH 4 HCO 3 . The fragments were reduced in 5 µl

45 mM dithiothreitol (DTT) and then incubated at 50 °C in

15 minutes. The solution was then cooled to room temperature, and the fragments were alkylated by adding 5 µl of 100 mM iodoacetamide (Sigma, St. Louis, MO). After 15 minutes of incubation at room temperature, the sample was diluted with 187 µl Milli

Q-water to a final urea concentration of 2.0 M. Trypsin (Worthington, Freehold, NJ) was then added at a 1:25 (w:w) ratio of enzyme to protein, and the protein was digested for 24 h at 37 °C. The digestion was terminated with the addition of 30 ul of TFA.

The protein fragments were then separated by high-performance liquid chromatography (HPLC) on a Waters 625 LC system (Millipore, Milford, MA) using a 2.1 x 250 mm,

5 micron Vydac C-18 column (Vydac, Hesperia, CA) equilibrated in 0.05% TFA and HPLC water (buffer A). The peptides were eluted with increasing concentration of 80% acetonitrile in 0.04% TFA (buffer B) with a gradient of 38-75% buffer B for 65-95 minutes and 75-98% buffer B for 95-105 minutes. Peptides were fractionated at a flow rate of 0.2 ml/min and detected at 210 nm.

After fractionation, the amino acid sequence of the peptides was analyzed by automated Edman digestion performed on an Applied Biosystems model 473A protein sequencer using the manufacturer's standard cycles and the model 610A data analysis software program, version 1.2.1. All sequencing reagents were supplied by Applied Biosystems. The amino acid sequences for 7 of the 8 internal fragments are shown below, where "X" indicates that the identity of the amino acid was not certain.

Primerout shaping

One of the internal amino acid sequences (shown in SEQ ID NO: 22) was then used to design a fully degenerate oligonucleotide primer, designated p4(R), as shown in SEQ ID NO. NO. 23.-

Example 4

PCR cloning of a canine Q-tmi fragment

The 5<1> part of the dog a™i gene was amplified from double-stranded dog spleen cDNA by PCR.

A. Formation of double-stranded canine spleen cDNA

1 g frozen material of a spleen from a juvenile dog was ground in liquid nitrogen on dry ice and homogenized in 20 ml RNA-Stat 60 buffer (Tel-Test B, Inc., Friendswood, TX). 4 ml of chloroform was added and the solution was extracted by centrifugation at 12,000 g for 15 minutes. RNA was precipitated from the aqueous layer with 10 ml of ethanol. Poly A<+>RNA was then selected on Dynal Oligo dT Dynabeads (Dynal, Oslo, Norway).

5 aliquots of 100 µg total RNA were combined and diluted with an equal volume of 2x binding buffer (20 mM Tris-HCl, pH 7.5, 1.0 M LiCl, 1 mM EDTA, 0.1% SDS). RNA was then incubated for 5 min with Oligo dT Dynabeads (1.0 ml or 5 mg of the microbeads for all samples). The microbeads were washed with buffer containing 10 mM Tris-HCl, pH 7.5, 0.15 M LiCl, -1 mM EDTA and 0.1% SDS, according to the manufacturer's suggested protocol, before elution of poly A<+>mRNA with 2 mM EDTA, pH 7.5. Double-stranded cDNA was then generated using the eluted poly A<+>mRNA and the Boehringer Mannheim cDNA synthesis kit in accordance with the manufacturer's suggested protocol.

B. Isolation of a partial dog poop cDNA

Oligonucleotide primers 5'Deg (SEQ ID NO: 9) and p4(R)

(SEQ ID NO: 23) was used in a standard PCR reaction using 150 ng of double-stranded cDNA, 500 ng of each primer, 200 uM dNTP and 1.5 units of Tag polymerase (Boehringer Mannheim) in Tag buffer (Boehringer Mannheim) with magnesium. The resulting products (1 µl of the original reaction) were subjected to a second round of PCR with the same primers to increase the product yield. This band was eluted from a 1% agarose gel on Na45 paper from Schleicher & Schuell (Keene, NH) in a buffer containing 10 mM Tris-HCl, pH 8, 1 mM EDTA, 1.5 M NaCl, at 65 °C, precipitated and ligated into the pCR<tm>II vector (Invitrogen, San

Diego, CA), using the TA cloning kit (Invitrogen) and the manufacturer's suggested protocol. The ligation mixture was transferred by electroporation into XL-1 blue bacteria (Stratagene). One clone, 2.7, was determined to contain sequences corresponding to axMi peptide sequences that were not used in the design of the primers.

Sequencing was performed with an Applied Biosystems 373A DNA sequencer (Foster City, CA), with a color deoxyterminator cycle sequencing (ABI) kit, in which fluorescently labeled dNTPs were incorporated into an asymmetric PCR reaction [McCabe, "Production of Single Stranded DNA by Asymmetric PCR", in PCR Protocols: A Guide to Methods and Applications, Innis et al. (ed.), pp. 7.6-83, Academic Press: New York (1990)] as follows. Samples were held at 96°C for 4 minutes and subjected to 25 cycles of the stepwise sequence: 96°C for 15 seconds; 50 °C for 1 second; 60 °C for 4 minutes. Sequence data were automatically loaded onto computer sample files, which included chromatograms and text files. The sequence of the entire insert in clone 2.7 is shown in SEQ ID NO. NO. 24.

Attempts to isolate the unabridged canine - Otmi ~ cDNA from the Stratagene library (as described in Example 2) were unsuccessful. Approximately 1 x 10<6> phage plaques were screened by hybridization under low stringency conditions using 30% formamide with clone 2.7 as a probe, but this yielded no positive clones. Attempts to amplify relevant sequences downstream from the sequences shown in clone 2.7 using specific oligonucleotides derived from clone 2.7, or degenerate primers based on amino acid sequence from other peptide fragments paired with a degenerate oligonucleotide based on the conserved α-subunit amino acid motif GFFKR [Tamura et al., supra], was also unsuccessful.

Example 5

Cloning of a putative human homologue of canine o-tmi

As an attempt to isolate a human sequence homologue

for the dog arm it was approx. 1 kb dog a™ fragment from clone 2.7 used as a probe. The probe was generated by PCR under conditions described in Example 2 using NT2 (as shown in SEQ ID NO: 25) and p4(R) primers (SEQ ID NO: 23).

5'-GTNTTYCARGARGAYGG-3' (SEQ ID NO: 25)

The PCR product was purified using the Qiagen (Chatsworth, GA) Quick Spin kit and the manufacturer's suggested protocol. The purified DNA (200 ng) was labeled with 200 uCi a<32->PdCTP using the Boehringer Mannheim "Random Prime" labeling kit

and the manufacturer's proposed protocol. Unincorporated isotope was removed using Sephadex G-25 (fine) gravity chromato-

graphics. The probe was denatured with 0.2 N NaOH and neutralized with 0.4 mM Tris-HCl, pH 8.0, before use.

Colony pick-up on Hybond filters (Amersham) of a human spleen cDNA library in pCDNA/Amp (Invitrogen, San Diego, CA) was prepared. The filters were first denatured and neutralized as described in example 2 and then incubated in a prehybridization solution (8 ml/filter) with 30% formamide at 50°C with gentle stirring for 2 hours. Labeled probe, as described above, was added to this solution and incubated with the filters for 14 hours at 42°C. The filters were washed twice in 2x SSC/0.1% SDS at 37°C and twice in 2x SSC/0.1% SDS at 50°C. The last rigorous washings were

1x SSC/0.1% SDS, twice at 65 °C (1x SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.0). The filters were exposed with Kodak X-Omat AR film for 6 h with an intensifying screen. Colonies giving signals on two different pull-ups were streaked onto plates of LB medium with magnesium (LBM)/carbenicillin and incubated overnight at 37°C. Resulting streaked colonies were picked up with Hybond filters, and these filters were processed as described above. The filters were hybridized under more stringent conditions with the 1 kb probe from clone 2.7, labeled as described above, in a 50% form-amide hybridization solution at 50°C for 3 h. Probed filters were washed with a final stringency of 0.1 x SSC/0.1% SDS at 65 °C and exposed with Kodak X-Omat AR film in

2.5 hours at -80 °C with an intensification screen. Positive colonies were identified and grown in LBM/carbenicillin medium overnight. DNA from the cultures was prepared using the Promega Wizard miniprep kit in accordance with the manufacturer's suggested protocol, and the resulting DNA was sequenced.

The initial screening resulted in 18 positive clones, while the second screening under more stringent hybridization conditions produced one positive clone designated 19A2. The DNA and the deduced amino acid sequences of the human α<i clone 19A2 are respectively shown in SEQ ID NO. NO. 1 and 2.

Characteristic properties of the human Od cDNA and the calculated polypeptide

Clone 19A2 comprises the entire coding region of the mature protein, plus 48 bases (16 amino acid residues) of the 5'-upstream signal sequence and 241 bases of 3'-untranslated sequence that do not terminate in a polyadenylation sequence. The core molecular weight of the mature protein is calculated to be approx. 125 kD. The extracellular domain is calculated to comprise approx. amino acid residues 17-1108 in SEQ ID NO. NO. 2. This extracellular area borders up to an approx. 20 amino acid region that is homologous to the human CD11c transmembrane region (residues 1109-1128 in SEQ ID NO: 2). The cytoplasmic domain comprises approx. 30 amino acids (approx. residues 1129-1161 in SEQ ID NO: 2). The protein also contains an area (around residues 150-352) with approx. 202 amino acids homologous to the I (insertion) domain common to CDlla, CDllb and CDllc [Larson and Springer, supra], aE [Shaw et al., J. Biol. Chem., 265:6016-6025 (1994)] and in VLA-1 and VLA-2 [Tamura et al., supra]. The I domain in other integrins has been shown to participate in ICAM binding [Landis et al., J. Cell. Biol., 120:1519-1527 (1993); Diamond et al., J. Cell. Biol., 120:1031-1043 (1993)], suggesting that eid can also bind members of the ICAM family of surface molecules. This region has not been shown to exist in any other integrin subunits.

The deduced amino acid sequence of ad shows approx. 36% identity with the sequence of CDlla, approx. 60% identity with CDllb and approx. 66% identity with CDllc. An alignment of amino acid sequences for CDllb (SEQ ID NO: 3), CDllc (SEQ ID NO: 4) and dd (SEQ ID NO: 2) is shown in Figure 1.

The cytoplasmic domains of α-subunits in β2 integrins typically differ from each other within the same species, while individual α-subunits show a high degree of homology across species boundaries. Consistent with these observations, the cytoplasmic region of dd is markedly different from CDlla, CDllb and CDllc, with the exception of a membrane-proximal GFFKR amino acid sequence that has been shown to be conserved among all α-integrins [Rojiani et al., Biochemistry , 30:9859-9866 (1991)]. Because the cytoplasmic tail region of integrins has been implicated in "inside out" signaling and avidity regulation [Landis et al., supra], it is possible that it interacts with cytosolic molecules different from those that interact with CDlla, CDllb and CDllc and as a result participates in signaling pathways that are different from those that

involving other β2 integrins.

The extracellular domain of da contains a conserved DGSGS amino acid sequence adjacent to the I domain; in CDllb, the DGSGS sequence is a metal-binding region required for ligand interaction [Michishita et al., Cell, 72:857-867 (1993)]. Three additional putative cation binding sites in CD11b and CD11c are conserved in the aa sequence at amino acids 465-474, 518-527 and 592-600 in clone 19A2 (SEQ ID NO: 1). The dd-I domain is 36%, 62%, and 57% identical to the corresponding regions in CDlla, CDllb, and CDllc, respectively, and the relatively low sequence homology in this region suggests that aa may interact with an additional set of extracellular proteins that are different from proteins with which other known β2 integrins react. Alternatively, the affinity of aa for known Pa-int egrin ligands, e.g. ICAM-1, ICAM-2 and/or ICAM-R, be different from the affinity demonstrated for the other β2 integrin/ICAM interactions [see Example 12].

Isolation of additional human cta cDNA clones for sequence confirmation

To confirm the DNA sequence encoding human Oa, additional human cDNAs were isolated by hybridization from a human spleen oligo-dt-primed cDNA library (Invitrogen) in pcDNA/Amp (described in Example 5), which was size-selected by agarose gel electrophoresis for cDNA with a length greater than 3 kb. The probe for hybridization was derived from a 5' region of aa, as described below. Hybridization conditions were the same as described above for isolation of the original human aa clone, except that after hybridization the filters were washed twice in 2 x SSC/0.1% SDS at room temperature and once in 2 x SSC/0, 1% SDS at 42 °C. The filters were exposed with Kodak X-Omat AR film overnight.

The 5'-da hybridization probe was generated using

PCR from the 19A2 clone using primers CD11c-5'For (SEQ ID NO: 94) and CD11c-5'Rev (SEQ ID NO: 95) under the following conditions. Samples were held at 94°C for 4 minutes and subjected to 30 cycles of the temperature step sequence i) 94°C, 15 seconds; ii) 5 °C, 30 seconds; and iii)

72 °C, 1 minute, in a Perkin-Elmer 9600 thermocycler.

The amplification product was purified using the BioRad (Hercules, CA) Prep-A gene kit in accordance with the manufacturer's suggested protocol. The resulting 5'-Od probe had a length of approx. 720 bases, which corresponds to the area from nucleotide 1121 to nucleotide 1839 in SEQ ID NO. NO. 1. The purified DNA (approximately 50 ng) was labeled with <32>P-dCTP using a Boehringer Mannheim (Indianapolis, Indiana) "Random Prime" labeling kit in accordance with the manufacturer's suggested protocol. Unincorporated isotope was removed using Centrisep Spin columns (Princeton Separations, Adelphia, NJ) in accordance with the manufacturer's suggested protocol. Labeled probe was added to the filters in a pre-hybridization solution containing 45% formamide, and incubation was allowed to proceed overnight at 50°C. After incubation, the filters were washed as described above. 13 colonies gave signals of duplicate uptake. Positive colonies were picked from master plates, diluted in LBM and carbenicillin (100 µg/ml) and spread in varying dilutions on Hybond filters (Amersham). Duplicate filters were hybridized with the same solution from the primary hybridization, and after hybridization the filters were washed with a final stringency of 2 x SSC/0.1% SDS at 42°C and exposed to film. 10 of the initially identified 13 positive colonies were confirmed in the second screening. Of these 10 clones, 2 (designated A7.Q and A8.Q) were sequenced and determined to encode human Od. Clone A7.Q was found to have a length of approx. 2.5 kb, including a 5' leader, part of a coding region and an additional 60 bases of 5' untranslated sequence.

The incomplete coding region was determined to have resulted from an aberrantly spliced intron region at the corresponding nucleotide 2152 in SEQ ID NO. NO. 1. Clone A8.Q was determined to have a length of approx. 4 kb, spanned the entire Od coding region and also included an intron sequence at the corresponding base 305 of SEQ ID NO:1. NO. 1. Compared to the originally isolated Od clone {SEQ ID NO. NO. 1) one difference was observed in that both the A7.Q and A8.Q clones were determined to contain a 3 base CAG codon insertion at base 1495. Sequences for clones A7.Q and A8.Q are shown in respectively

SEQUENCE ID. NO. 96 and 97, and a composite human sequence derived from clones A7.Q and A8.Q and its corresponding deduced amino acid sequence are shown in SEQ ID NO:1, respectively. NO. 98 and 99.

Example 6

Northern analysis of human aa expression in tissues

To determine the relative expression level and tissue specificity of ctd, Northern analysis was performed using fragments from clone 19A2 as probes. Approximately 10 µg of total RNA from each of several human tissues or cultured cell lines was applied to a formaldehyde agarose gel in the presence of 1 µg of ethidium bromide. After electrophoresis at 100 V for 4 h, RNA was transferred to a nitrocellulose membrane (Schleicher & Schuell)

by immersion in 10 x SSC overnight. The membrane was heated for 1.5 hours at 80 °C under vacuum. Prehybridization solution containing 50% formamide in 3-(N-morpholino)propanesulfonic acid {MOPS) buffer was used to block the membrane for 3 h

at 42 °C. Fragments of clone 19A2 were labeled with the Boehringer Mannheim "Random Prime" kit according to the manufacturer's instructions, including both aP<32>dCTP and aP<32>dTTP. Unincorporated label was removed on a Sephadex G-25 column in TE buffer. The membrane was probed with 1.5 x IO<6> counts per ml prehybridization buffer. The blot was then washed successively with 2 x SSC/0.1% SDS at room temperature, 2 x SSC/0.1%

SDS at 42 °C, 2 x SSC/0.1% SDS at 50 °C, 1 x SSC/0.1% SDS

at 50 °C, 0.5 x SSC/0.1% SDS at 50 °C and 0.1 x SSC/0.1% SDS

at 50 °C. The blot was then exposed with film for 19 hours.

Hybridization using a BstXI fragment from clone 19A2 (corresponding to nucleotides 2011-3388 in SEQ ID NO: 1) revealed a weak signal in the ca. The 5 kb range in total RNA from liver, placenta, thymus and tonsil. No signal was detected in samples from kidney, brain or heart. The amount of RNA present in the kidney field was minimal, as determined by ethidium bromide staining.

Using a second fragment of clone 19A2 (comprising the range from bases 500 to 2100 of SEQ ID NO: 1), RNA transcripts of two different sizes were detected in a human multi-tissue Northern (MTN) blot using poly A<+->RNA (Clontech). An approx. A 6.5 kb band was observed in spleen and skeletal muscle, while a 4.5 kb band was detected in lung and peripheral blood leukocytes. The variation in observed sizes could be caused by tissue-specific polyadenylation, cross-reactivity of the probe with other integrin family members, or hybridization with alternatively spliced mRNAs.

Northern analysis using a third fragment from clone 19A2, spanning nucleotides 2000-3100 of SEQ ID NO. NO. 1, gave results consistent with the results using the other clone 19A2 fragments.

RNA from three cell lines of the myeloid lineage was also probed using the fragments corresponding to nucleotides 500-2100 and 2000-3100 in SEQ ID NO. NO. 1. A THP-1 cell line, previously stimulated with PMA, gave a diffuse signal in the same size range (about 5.0 kb) with a slightly stronger intensity than the tissue signals. RNA from unstimulated and DMSO-stimulated HL-60 cells hybridized with the Od probe with the same intensity as the tissue samples, however, PMA treatment appeared to increase signal intensity. Because PMA and DMSO direct HL60 cell differentiation toward monocyte/macrophage and granulocyte pathways, respectively, this result suggests elevated Od expression in monocyte/macrophage cell types. U937 cells expressed the Od-bud cabinet, and this signal did not increase with PMA stimulation. No band was detected in Molt, Daudi, H9, JY or Jurkat cells.

Example 7

Transient expression of human dg constructs

A. Formation of expression constructions

The human clone 19A2 lacks an initiation methionine codon" and possibly some of the 51 signal sequence. Therefore, to generate a human expression plasmid containing 19A2 sequences, two different strategies were used. In the first, two plasmids were constructed in which signal peptide sequences derived from genes encoding either CD11b or CD11c, was spliced into clone 19A2 to form a chimeric Od sequence.In the second method, a third plasmid was constructed in which an adenosine base was added at position 0 in clone 19A2 to encode an initiating methionine.

The three plasmids contained different regions encoding the 5<1> part of the Od sequence or the chimeric Ad sequence. The dd region was PCR amplified {see conditions in Example 2) with a specific 3' primer, BåmRev (shown below in SEQ ID NO: 26), and one of three 5' primers. The three 5' primers contained, in order: (1) identical non-specific bases at positions 1-6 allowing cleavage, an EcoRI site from positions 7 to 12 and a consensus Kozak sequence from positions 13 to 18; (2) part of the CDllb (primer ER1B) or CDllc (primer ERIC) signal sequence or an adenosine (primer ER1D), and (3) an additional 15-17 bases that specifically overlap 5' sequences from clone 19A2 for to allow primer annealing. Primer ER1B, ERIC or ER1D are respectively shown in SEQ ID NO. NO. 27, 28 or 29, where the initiating methionine codon is underlined and the EcoRI site is double underlined.

The resulting PCR product was digested with EcoRI and BamHI.

All three plasmids contained a common second Od region (to be inserted immediately downstream of the 5' region described in the previous section) including the 3<1> end of the Od clone. The second Od region, extending from nucleotide 625 into the XbaI site in the vector 3' polylinker region of clone 19A2, was isolated by digestion of clone 19A2 with BamHI and XbaI.

Three ligation reactions were performed in which the 3'-Od-BamHI/XbaI fragment was ligated to one of the three S^-ctd-Eco-RI/BamHI fragments using Boehringer Mannheim ligase buffer and T4 ligase (1 unit per .reaction). After incubation for 4 hours at 14°C, an appropriate amount of vector pcDNA.3 (Invitrogen) digested with EcoRI and XbaI was added to each reaction mixture with an additional unit of ligase. The reactions were allowed to proceed for an additional 14 hours. One tenth of the reaction mixture was then transferred to competent XL-1-Blue cells. The resulting colonies were grown, and DNA was isolated as described in Example 5. Digestion with BcoRI identified three clones positive for this restriction site and thus signal sequences were constructed. The clones were designated pATM.B1 (CD11b/dd, from primer ER1B), pATM.CIO (CD11c/ad, from primer ERIC) and pATM.D12 (adenosine/cid, from primer ER1D). The presence of the appropriate signal sequences in each clone was verified by nucleic acid sequencing.

B. Transfection of COS cells

Expression of the ad plasmids discussed above was effected by cotransfection of COS cells with the individual plasmids and a CD18 expression plasmid, pRC.CD18. As a positive control, COS cells were also cotransfected with the plasmid pRC.CD18 and a CDlla expression plasmid, pDC.CDllA.

Cells were transferred in culture medium (DMEM/10% FBS/penstrep) into 10 cm Corning tissue culture-treated Petri dishes at 50% confluence 16 h before transfection. Cells were removed from the plates with Versene buffer (0.5 mM NaEDTA in PBS) without trypain in all procedures. Before transfection, plates were washed once with serum-free DMEM. 15 µg of each plasmid was added to 5 ml of transfection buffer (DMEM with 20 µg/ml DEAE-dextran and 0.5 mM chloroquine) on each plate. After incubation for 1.5 h at 37 °C, the cells were shocked for 1 min with 5 ml DMEM/10% DMSO. This DMSO solution was then replaced with 10 ml/plate culture medium.

Resulting transfectants were analyzed by ELISA, FACS and immunoprecipitation, as described in Examples 8, 9 and 10.

Example 8

ELISA analysis of COS transfectants

To determine whether the COS cells cotransfected with CD18 expression plasmid pRC.CD18 and whether an oa plasmid expressed aa on the cell surface in association with CD18, ELISA was performed using primary antibodies against CD18 (e.g. TS1/18 purified from ATCC HB203). As a positive control, ELISA was also performed on cells cotransfected with the CD18 expression plasmid and a CDlla expression plasmid, pDC.CDllA. The primary antibodies in this control included CD18 antibodies and anti-CDlla antibodies (eg, TS1/22 purified from ATCC HB202).

For ELISA, cells from each plate were removed with Versene buffer and transferred to a single 96-well flat-bottom Corning tissue culture plate. Cells were allowed to incubate in culture medium for 2 days before analysis. The plates were then washed twice with 150 µl/well D-PBS/0.5% teleost skin gelatin solution (Sigma). This buffer was used in all steps except during development. All washings and incubations were performed at room temperature. The wells were blocked with gelatin solution for 1 hour. Primary antibodies were diluted to 10 µg/ml in gelatin solution, and 50 µl was then added to each well. Three wells were then used for each primary antibody. After incubation for 1 hour, the plates were washed three times with 150 µl/well gelatin solution. Secondary antibody (goat anti-mouse Ig/HRP-Fc-specific [Jackson, West Grove, PA]) at a dilution of 1:3500 was added in an amount of 50 µl/well, and the plates were incubated for 1 hour . After three washes, plates were developed for 20 min with 100 µl/well o-phenyldiamine solution (OPD) (Sigma) (1 mg/ml OPD in citrate buffer) before addition of 50 µl/well 15% sulfuric acid.

Analysis of transfectants in the ELISA format with anti-CD18 specific antibodies revealed no significant expression above the background level in cells transfected only with the plasmid encoding CD18. Cells co-transfected with plasmid containing CDlla and CD18 showed an increase in expression compared to the background when analyzed with CD18-specific antibodies or with reagents specific for CDlla. Further analysis of cells cotransfected with plasmids encoding CD18 and one of the aa expression constructs (pATM.ClO or pATM.D12) revealed that cell surface expression of CD18 was rescued by concomitant expression of dd. The increase in detectable CD18 expression in COS cells transfected with pATM.ClO or pATM.D12 was comparable to that observed in cotransfected CD11a/CD18-positive control cells.

Example 9

FACS analysis of COS transfectants

For FACS analysis, cells in Petri dishes were added with fresh culture medium the day after transfection and incubated for 2 days before analysis. Transfected cells were removed from the plates with 3 ml Versene, washed once with 5 ml FACS buffer (DMEM/2% FBS/0.2% sodium azide) and diluted to 500,000 cells/sample in 0.1 ml FACS buffer. 10 µl of either 1 mg/ml FITC-conjugated CD18, CDlla, or CDllb-specific antibodies (Becton Dickinson) or 800 µg/ml CFSE-conjugated mouse 23F2G (anti-CD18) (ATCC HB11081) were added to each sample . The samples were then incubated on ice for 45 min, washed three times with 5 ml/wash FACS buffer and resuspended in 0.2 ml FACS buffer. The samples were processed on the Becton Dickinson FACScan, and the data were analyzed using Lysis II software (Becton Dickinson).

COS cells transfected only with CD18 sequences did not stain for CD18, CD11a, or CD11b. When they were cotransfected with CDlla/CD18, approx. 15% of the cells stained with antibodies against CDlla or CD18. All cells transfected with CD18 and any ad construct gave no detectable staining for CDlla and CDllb. The pATM.B1, pATM.ClO and pATM.Dl2 groups stained 4%, 13% and 8% positive for CD18, respectively. Fluorescence in the positive population in the CDlla/CD18 group was four times higher than the background. In comparison, the cotransfection of ad constructs with the CD18 construct produced a positive population that showed a 4-7 fold increase in fluorescence intensity relative to background.

Example 10

Biotin-labeled immunoprecipitation of human ad/CDl8 complexes from cotransfected COS cells

Immunoprecipitation was attempted on cells cotransfected with CD18 and each of the Od expression plasmids separately described in Example 7, to determine whether α& could be isolated as part of the α{J-heterodimeric complex characteristic of integrins.

Transfected cells (1-3 x 10<8> cells/group) were removed from Petri dishes with Versene buffer and washed three times in 50 ml/group D-PBS. Each sample was labeled with 2 mg of sulfo-NHS-biotin (Pierce, Rockford, IL) for 15 min at room temperature. The reaction was stopped by washing three times in 50 ml/sample cold D-PBS. Washed cells were resuspended in 1 ml of lysis buffer (1% NP40; 50 mM Tris-HCl, pH 8.0; 0.2 M NaCl;

2 mM Ca<++>; 2 mM Mg<++> and protease inhibitors) and incubated in

15 minutes on ice. Insoluble material was pelleted by centrifugation at 10,000 g for 5 min, and the supernatant was transferred to fresh tubes. To remove the material that was non-specifically reactive with mouse immunoglobulin, a pre-clearing step was first performed. 25 µg mouse immunoglobulin (Cappel, West Chester, PA) was incubated with supernatants at 4°C.

After 2.5 hours, 100 µl (25 µg) of rabbit anti-mouse Ig-conjugated Sepharose (prepared from protein A-Sepharose 4B and rabbit anti-mouse IgG, both from Zymed, San Francisco, CA) was added to each sample ; incubation was continued at 4°C with rocking for 16 hours. Sepharose beads were removed from the supernatant by centrifugation. After pre-clearing, the supernatants were then treated with 20 µg of anti-CD18 antibody (TS1.18) for 2 h at 4 °C. Antibody/antigen complexes were isolated from supernatants by incubation with 100 µl/sample of the rabbit anti-mouse/protein A-Sepharose preparation described above. The beads were washed four times with 10 mM HEPES, 0.2 M NaCl, and 1% Triton-X-100. Washed beads were pelleted and boiled for 10 minutes in 20 µl of 2x Laemmli sample buffer with 2% mercaptoethanol. Samples were centrifuged and chromatographed on an 8% preloaded Novex polyacrylamide gel (Novex) at 100 V for 30 min. Protein was transferred to nitrocellulose membranes (Schleicher & Schuell) in TBS-T buffer at 200 mA for 1 h. Membranes were blocked for 2 h with 3% BSA in TBS-T. The membranes were treated with 1:6000 dilution of streptavidin-horseradish peroxidase (POD) (Boehringer Mannheim) for 1 h, followed by three washes in TBS-T. Amersham enhanced chemiluminescence kit was then used according to the manufacturer's instructions to develop the blot. The membrane was exposed with hyperfilm MP (Amersham) for 0.5-2 minutes.

Immunoprecipitation of CD18 complexes from cells transfected with pRC.CD18 and either pATM.B1, pATM.ClO or pATM.D12 revealed surface expression of a heterodimeric type consisting of approx. 100 kD p-chain, which is consistent with the predicted size of CD18, and an a-chain of approx. 150 kD which corresponds to aa.

Example 11

Stable transfection of human aa in Chinese hamster ovary cells

To determine whether aa is expressed on the cell surface as a heterodimer in association with CD18, cDNA encoding each chain, both transiently and stably, was transfected into a cell line lacking both cia and CD18.

For these experiments, the ad cDNA was extended with additional leader sequences and a Kozak consensus sequence, as described in Example 7, and subcloned into expression vector pcDNA3. The final construct, designated pATM.D12, was cotransfected with a modified commercial vector, pDCl.CDIS, encoding human CD18, into dihydrofolate reductase-(DHFR) "-Chinese hamster ovary (CHO) cells. The plasmid pDCl.CDIS encodes a DHFR<+-> marker, and transfectants can be selected using an appropriate nucleoside-deficient medium.The modifications resulting in pDCl.CDIS are as follows.

The plasmid pRC/CMV (Invitrogen) is a mammalian expression vector with a cytomegalovirus promoter and an ampicillin resistance marker gene. A DHFR gene from the plasmid pSC1190-DHFR was inserted into pRC/CMV-5' for the SV40 origin of replication. A polylinker from the 5<1> region of the plasmid pHF2G-DHF was also ligated into the pRC/CMV/DHFR construct, 3' to the DHFR gene. The CD18 coding sequences are then cloned into the resulting plasmid between the 5<1> flanking polylinker region and the bovine growth hormone poly A coding region.

Surface expression of CD18 was analyzed by flow cytometry using the monoclonal antibody TS1/18. Heterodimer formation detected between α<j and CD18 in this cell was consistent with the immunoprecipitation described in Example 10 with transient expression in COS cells.

Example 12

Human aa binds to ICAM-R in a CD18-dependent manner

In view of reports demonstrating interactions between the leukocyte integrins and the intercellular adhesion molecules (ICAMs) that mediate cell-cell contact [Hynes, Cell, 69:11-25 (1992)], the ability of CHO cells expressing Od/CD18 to to bind ICAM-1, ICAM-R or VCAM-1, determined using two methods.

In repeated assays, soluble ICAM-1, ICAM-R, or VCAM-1-IgG1 fusion proteins were immobilized on plastic, and the ability of aa/CD18-CHO-transfected cells to bind the immobilized ligand was determined. Transfected cells were labeled internally with calcein, washed in binding buffer (RPMI with 1% BSA) and incubated in either buffer alone (with or without

10 ng/ml PMA) or buffer with anti-CD18 monoclonal antibodies at 10 ug/ral. Transfected cells were added to 96-well Iramulon 4 microtiter plates previously coated with soluble ICAM-1/IgGl, ICAM-R/IgGl, or VCAM-1/IgGl fusion protein or bovine serum albumin (BSA) as a negative control. Design of the soluble forms of these adhesion molecules is described and fully explained in US Patent Application No. 08/102,852, filed August 5, 1993. Wells were blocked with 1% BSA in PBS prior to addition of labeled cells. After washing the plates by immersion in PBS with 0.1% BSA for 20 minutes, residual total fluorescence in each well was measured using a Cytofluor 2300 (Millipore, Milford, MA).

In experiments with immobilized ICAM, dd/CD18 cotransfectants consistently showed a 3-5 fold increase in binding to ICAM-R/IgG1 wells relative to BSA-coated wells. The specificity and CD18 dependence of this binding was demonstrated by the inhibitory effects of anti-CD18 antibody TS1/18. The binding of cells transfected with CD11a/CD18 to ICAM-1/IgG1 wells was comparable to the binding observed with BSA-coated wells. CD11a/CD18-transfected cells showed a 2-3 fold increase in binding to ICAM-1/IgG1 wells only after pretreatment with PMA. PMA treatment of Od/CD18 transfectants did not affect binding to ICAM-1/IgG1 or ICAM-R/IgG1 wells. No detectable binding of ad/CD18 transfectants to VCAM-1/IgG1 wells was observed.

Binding of ad/CD18-transfected cells to soluble ICAM-1/IgG1, ICAM-R/IgG1 or VCAM-1/IgG1 fusion proteins was determined by flow cytometry. Approximately 1 million Gm/CDIB-transfected CHO cells (grown in rotary flasks for higher expression) per measurement was suspended in 100 µl binding buffer (RPMI and 1% BSA) with or without 10 µg/ml anti-CD18 antibody. After 20 minutes of incubation at room temperature, the cells were washed in binding buffer, and soluble ICAM-1/IgG1 or ICAM-R/IgG1 fusion protein was added to a final concentration of 5 µg/ml. Binding was allowed to occur for 30 minutes at 37°C, after which the cells were washed three times and resuspended in 100 µl binding buffer containing FITC-conjugated sheep antihuman IgG at a 1:100 dilution. After 30 min incubation, samples were washed three times and suspended in 200 µl binding buffer for analysis with Becton Dickinson FACScan.

Approximately 40-50% of the α<i/CD18 - transfectants indicated binding to ICAM-R/IgG1, but no binding to ICAM-1/IgG1 or VCAM-1/IgG1 proteins. Pretreatment of transfected cells with PMA has no effect on Oa/CD18 binding to either ICAM-1/IgG1, ICAM-R/IgG1, or VCAM-1/IgG1, which was consistent with the immobilized adhesion assay. Binding to ICAM-R was reduced to background levels after treatment of Od/CD18 transfectants with anti-CD18 antibody TS1/18.

The collective data from these two binding assays illustrate that Od/CD18 binds to ICAM-R preferentially compared to ICAM-1 and VCAM-1. The Od/CD18 binding preference for ICAM-R over ICAM-1 is in contrast to that observed with CD11a/CD18 and CD11b/CD18. Modulation of aa/CD18 binding can thus be expected to selectively affect normal and pathological immune function where ICAM-R plays a prominent role. Results from similar assays in which antibodies immunospecific for different extracellular domains of ICAM-R were tested for their ability to inhibit binding of ICAM-R to ctd/CD18 transfectants further indicated that cid/CD18 and CDlla/CD18 react with different domains of

ICAM-R.

The inability of CD11a/CD18 to bind ICAM-1/IgG1 or ICAM-R/IgG1 in solution suggests that the binding affinity between CD11a/CD18 and ICAM-1 or ICAM-R is too low to allow binding in solution. However, detection of aa/CD18 binding to ICAM-R/IgG1 suggests an exceptionally high binding affinity.

The FACS adhesion assay described above was used to test the binding of an ICAM-R mutant, E37A/Ig, to CHO cells expressing cid/CD18. E37A/Ig has been shown to prevent binding to an LFA-1/Ig chimera [Sadhu et al., Cell Adhesion and Communication, 2:429-440 (1994)]. The mutant protein was expressed in a soluble form from a stably transfected CHO cell line and purified through a Prosep-A column, as described by Sadhu et al., supra.

E37A/Ig binding with the ad/CD18 transfectants was not detected by repeated assays. The mean fluorescence intensity (MFI) of the E37A/Ig chimera detected using FITC-conjugated antihuman antibody was identical to the MFI of the detection antibody alone, indicating that there was no detectable signal above background using the E37A/Ig mutant protein in the analysis. In an ELISA, performed as described in Example 14, the E37A/lg mutant also did not appear to bind immobilized oa/CD18.

Q-d- binding to iC3b

Complement component C3 can be proteolytically cleaved to form the complex iC3b, which initiates the alternative pathway of complement activation and ultimately leads to cell-mediated destruction of a target. Both CDllb and CDllc have been implicated in iC3b binding and subsequent phagocytosis of iC3b-coated particles. A peptide fragment in the CD11b-I domain has recently been identified as the site of iC3b interaction [Ueda et al., Proe. Nati. Acad. Pollock. (USA), 51:10680-10684 (1994)]. The region of iC3b binding is highly conserved in CDllb, CDllc and eid, suggesting a dd/iC3b binding interaction.

Binding of Oa to iC3b is performed using transfectants or cell lines naturally expressing eid (eg, PMA-stimulated HL60 cells) and iC3b-coated ovine red blood cells (sRBC) in a rosette assay [Dana et al., J .Clin. Invest., 73:153-159 (1984)]. The ability of ad/CD18-CHO transfectants, VLA4-CHO transfectants (negative control) and PMA-stimulated HL60 cells (positive control) to form rosettes is compared in the presence and absence of anti-CD18 monoclonal antibody (e.g. . TS1/18.1).

Example 13

Screening by late-instillation proximity analysis_

Specific inhibitors of binding between the ad-ligands according to the invention and their binding partners (ad-ligand/antiligand pairs) can be determined in many different ways, such as by scintillation proximity analysis techniques, as generally described in US patent no. 4,271,139, Hart and Greenwald, Mol. Immunol., 12:265-267 (1979), and Hart and Greenwald, J. Nuc. Med., 20:1062-1065 (1979), all of which are incorporated herein by reference.

Briefly described, a member of the dd-ligand/anti-ligand pair is bound to a solid support material, either directly or indirectly. Indirect capture would involve a monoclonal antibody, directly bound to the support material, which recognizes a specific epitope at the C-terminus of the soluble integrin β-chain protein. This epitope will be either the hemagglutinin protein or the mycobacterial IIIE9 epitope [Anderson et al., J. Immunol., 141:607-613 (1988)]. A fluorescent agent is also bound to the support material. Alternatively, the fluorescent agent may be integrated into the solid support material, as described in US Patent No. 4,568,649, incorporated herein by reference. The member of the ad-ligand/anti-ligand pair that is not bound to the support material is labeled with a radioactive compound that emits radiation capable of exciting the fluorescent agent. When the ligand binds to the radiolabeled antiligand, the label is brought sufficiently close to the support material-bound fluorescent agent that it is excited and causes the emission of light. When unbound, the marker is generally too far from the solid support material to excite the fluorescent agent, and light emission is low. The emitted light is measured and correlated with binding between the ligand and the antiligand. Addition of a binding inhibitor to the sample will reduce the fluorescence emission by preventing the radioactive tracer from being captured in the vicinity of the solid support material. Binding inhibitors can therefore be identified by their effect on fluorescence emissions from the samples. Potential anti-ligands against Od can also be identified in a similar way.

The soluble recombinant cid/CD18-leucine "zipper" construct (see Example 14) is used in a scintillation proximity assay to screen for modulators of CAM binding using the following method. The recombinant integrin is immobilized with a non-blocking anti-α-subunit or anti-β-subunit antibody previously coated on a plate with embedded late-instillation material. Compounds from a chemical library and a specific biotinylated CAM/Ig chimera are simultaneously added to the plate. Binding of the CAM/Ig chimera is detected with labeled streptavidin. In the assay, ICAM-1/Ig and ICAM-3/Ig are biotinylated with NHS-sulfobiotin LC (long chain, Pierce) in accordance with the manufacturer's proposed protocol. Labeled proteins are still reactive with CAM-specific antibodies and can be shown to react with immobilized LFA-1 by ELISA, with detection by streptavidin-HRP and subsequent development with OPD.

Alternatively, the recombinant leucine "zipper" protein is purified, or partially purified, and coated directly on the plate with embedded late-instillation material. Unlabeled CAM/Ig chimeras and chemical library compounds are added simultaneously. Bound CAM/Ig is detected with 12<5>I-labeled antihuman Ig.

As a further alternative, purified CAM/Ig protein is immobilized on the scintillation plate. Chemical library compounds and concentrated supernatant from cells expressing recombinant leucine "zipper" integrin are added to the plate. Binding of the recombinant integrin is detected with a labeled, non-blocking α- or β-subunit antibody.

Example 14

Soluble human aa expression constructs

The expression of truncated, soluble human dd/CD18 heterodimer protein readily provides purified material for immunization and binding assays. The advantage of forming soluble protein is that it can be purified from supernatants rather than from cell lysates (as with unabridged membrane-bound Od/CD18); recycling is therefore improved, and impurities are reduced.

The soluble aa expression plasmid was constructed as follows. A nucleotide fragment corresponding to the range from bases 0 to 3161 in SEQ ID NO. NO. 1, cloned in plasmid pATM.D12, was isolated by digestion with HindIII and AatIII. A PCR fragment corresponding to bases 3130 to 3390 of SEQ ID NO. NO. 1, which overlaps Hindi 11 1 hat. II fragment and contains an additional ATluI restriction site at the 3'-terminus, was amplified from pATM.D12 with primers sHAD.5 and sHAD.3, shown in SEQ ID NO:1, respectively. NO. 30 and 31.

The PCR amplification product was digested with Aatll and M1ul and ligated to the HindIII/Aatll fragment. The resulting product was ligated into the Hindi11/MluI cleaved plasmid pDC1.s.

This construct is co-expressed with soluble CD18 in stably transfected CHO cells, and the expression is detected by autoradiographic visualization of immunoprecipitated CD18 complexes derived from <35>S-methionine-labeled cells. The construct is also co-expressed with CD18 in 293 cells [Berman et al., J. Cell. Biochem., 52:183-195 (1993)].

Soluble, unabridged aa construction

Alternative dd expression constructs are also encompassed by the invention. To facilitate expression and purification of an intact ad/CD18 heterodimer, soluble Od and CD18 expression plasmids will be constructed to include a "leucine zipper" fusion sequence that will stabilize the heterodimer during purification [Chang et al., Proe. Nati. Acad. Pollock. (USA), 51:11408-11412 (1994)]. Briefly, DNA coding for the acidic and basic amino acid strands of the zipper is formed by primer annealing using oligonucleotides, described by Chang et al. The DNA sequences have been further modified to include additional Aflul and XbaI restriction sites at the 5' and 3' ends of the DNA, respectively, to facilitate subcloning into previously described eid or CD18 expression constructs.

In addition, sequences representing either hemagglutinin protein or a polyhistidine sequence have been added, as well as a stop codon inserted after the XbaI site. The hemagglutinin or polyhistidine sequences are incorporated to facilitate affinity purification of the expressed protein. Sequences encoding the basic strand of the zipper are incorporated into the plasmid vector expressing CD18; the acidic strand is inserted into the α-chain structure. Upon expression of the modified aa and CD18 proteins in a host cell, it is believed that interaction between the acidic and basic strands of the zipper structure will stabilize the heterodimer and allow isolation of the intact cid/CD18 molecule by affinity purification, as described above.

Plasmids were constructed for expression of soluble o-d and CD18 with acidic and basic "leucine zipper" sequences and transfected into COS cells using the DEAE/dextran method described in Example 7. The resulting protein was referred to as ad/CD18LZ. Hemagglutinin and polyhistidine "tag patches" were not incorporated into Oa/CDISLZ. Transfected cells were cultured for 14 days under reduced serum {2%) conditions. Supernatants harvested every 5 days from transfected cells were analyzed for protein production by ELISA, as described in Example 8. Briefly, the Od/CD18LZ heterodimer was immobilized on plates coated with anti-Od monoclonal antibody 169B (see Example 15). The Od/CD18LZ complex was detected by the addition of a biotinylated anti-CD18 monoclonal antibody, TS1/18.1 (see Example 18), followed by the addition of streptavidin/horseradish peroxidase (HRP) conjugate and o-phenyldiamine (OPD). Protein was clearly detectable in the supernatants.

Binding assays using soluble, unabridged Qd expression products

Functional binding assays using the soluble, unabridged ad/CD18LZ heterodimer described above were performed by immobilizing the heterodimer on plates coated with monoclonal antibody 169B or a non-blocking anti-CD18 monoclonal antibody (see Example 15). Wells were blocked with fish skin gelatin to prevent non-specific binding before addition of CAM/Ig chimeras (see Example 12) at a starting concentration of 10 pg/ml. Binding of the chimeras to ad/CD18 was detected with a goat antihuman lg-HRP conjugate (Jackson Labs) and subsequent development with OPD.

VCAM-1/lg was observed to bind to captured cid/CD1SLZ in an amount 3-5 times higher than to captured CD11a/CD18. ICAM-1/Ig and ICAM-2/Ig bound soluble CD11a/CD18 heterodimers at an amount 15- and 10-fold greater than background, respectively, but did not bind cm/CD1S. VCAM-1 binding was reduced by approx. 50% in the presence of the VCAM-1-specific antibodies 130K and 130P used in combination.

The binding assay was also performed with the ICAM/Ig protein immobilized on 96-well plates, followed by the addition of recombinant soluble integrin to cell supernatant. Binding of the soluble integrins was detected with an unlabeled, non-blocking α- or β-subunit-specific mouse antibody, followed by incubation with HRP-conjugated goat anti-mouse antibody and development with OPD.

Results indicated that non-blocking antibody detected dd/CD18LZ binding to ICAM-R/Ig at an amount 10-fold greater than the binding detected in control wells without antibody. Soluble ad/CD18 binding was not detected with immobilized ICAM-1/Ig, however, binding was detected between Od/CD18 and immobilized CD11b/CD18 and CD11a/CD18 at an amount 15 and 5 times greater than background binding, respectively.

Because previous studies have shown that CDllb and CDllc bind lipopolysaccharide (LPS) [Wright, Curr. Opinion. Immunol., 3:83-90 (1991); Ingalls and Golenbock, J. Exp. Med., 181:1473-1479 (1995)], LPS binding to dd/CD18 was also determined using flow cytometry and plate-based assays. Results indicated that FITC-labeled LPS isolated from S. Minnesota and S. typhosa (both obtained from Sigma) by

20 pg/ml was able to weakly bind c 2 /CD1S-transfected CHO cells. No binding was observed with untransfected CHO control cells. In ELISA format assays, 0.5-3.0 pg bound biotinylated LPS [Luk et al., Alan. Biochem., 232:217-224

(1995)] immobilized α<i/CD18LZ with a signal fourfold greater than the capture antibody and blocking reagent alone. Apparent binding of LPS to CD11a/CD18 was excluded by subtracting background binding to anti-CD11a antibody TS2/4 from each experimental value.

To identify other ligands for Od/CD18, the recombinant ad/CD18LZ protein is used in a two-stage investigation. Binding of different cell types to immobilized protein is used to determine which cells express oa ligands on the cell surface. Antibody inhibition is then used to determine whether the observed cell binding results from interaction with known surface adhesion molecules. If no inhibition occurs, co-immunoprecipitation with a<j/CD18LZ bound to proteins from lysates of cells that will bind a<j is used to try to identify the ligand.

Soluble human aa-I domain expression constructs

It has previously been reported that the I domain of CDlla can be expressed as an independent structural unit that maintains ligand binding capabilities and antibody recognition [Randi and Hogg, J. Biol. Chem., 269:12395-12398 (1994); Zhou et al., J. Biol. Chem., 269:17075-17079 (1994); Michishita et al., Cell, 72:857-867 (1993)]. To form a soluble fusion protein comprising the Oa-I domain and human IgG4, the α-I domain is amplified by PCR using primers designed to add flanking BamHI and XhoI restriction sites to facilitate subcloning. These primers are shown in SEQ ID NO. NO. 32 and 33, where restricted seats are underlined. The C nucleotide immediately 3' of the BamHI site in SEQ ID NO. NO. 32 corresponds to nucleotide 435 in SEQ ID NO. NO. 1; The G-nucleotide 3' of the XhoI site in SEQ ID NO. NO. 33 is complementary to nucleotide 1067 in SEQ ID NO. NO. 1. The amplified I domain is cleaved with the appropriate enzymes, the purified fragment is ligated into the mammalian expression vector pDC, and the prokaryotic expression vector pGEX-4T-3 (Pharmacia) and the I domain fragment are sequenced. The fusion protein is then expressed in COS, CHO or E. coli cells transfected or transformed with an appropriate expression construct.

If the affinity of ad towards ICAM-R is given, expression of the ad-I domain may have sufficient affinity to be a useful inhibitor of cell adhesion in which eid participates.

Analysis of human ad-I domain/IgG4 fusion proteins

Protein was separated by SDS-PAGE under reducing and non-reducing conditions and visualized by either silver staining or Coomassie staining. Protein was then transferred to Immobilon PVDF membranes and subjected to Western blot analysis using antihuman IgG monoclonal antibodies or antibovine Ig monoclonal antibodies.

Detected protein was determined to migrate at approx.

120 kD under non-reducing conditions, and at approx. 45 kD under reducing conditions. Smaller bands were also detected on non-reducing gels at ca. 40-50 kD, and these bands were reactive with the antihuman, but not antibovine, antibodies. A minor band of 200 kD was determined to be bovine lg by Western blot.

Binding assays using I domain expression products The ability of the I domain to specifically recognize ICAM-R/IgG chimeric protein was tested in an ELISA format. Serial dilutions of Od-I domain-IgG4 fusion protein (Io.d/IgG4) in TBS were incubated with ICAM-l/IgGm, ICAM-R/IgG, VCAM-l/IgG or an irrelevant IgGl myeloma protein immobilized on Immulon IV RIA/EIA plates. CDlla-I domain/IgG chimeric protein and human IgG4/kappa myeloma protein were used as negative controls. Bound IgG4 was detected with the biotinylated anti-IgG4 monoclonal antibody HP6023, followed by the addition of streptavidin peroxidase conjugate and development with the substrate o-phenyldiamine.

In repeated analyses, no binding of the CDlla/IgG4 protein or the IgG4 myeloma protein to any of the immobilized proteins was detected. The Iad/IgG4 protein did not bind to fish skin gelatin or bovine serum albumin blocking agents, human IgG1 or ICAM-1/IgG. A 2-3 fold increase of the binding signal compared to the background was detected in ICAM-R/IgG protein coated wells using 1-5 pg/ml concentrations of Icid/IgG4 protein. The signal in VCAM-1/IgG protein-coated wells was 7-10 times higher than the background. In previous analyses, ad/CD18-transfected CHO cells did not bind VCAM-1/IgG protein, suggesting that VCAM-1 binding may be characteristic of isolated I-domain amino acid sequences.

Additional Qd-1 domain constructs

Additional eid-1 domain constructs are formed in the same way as the preceding construct, but by incorporating more amino acids around the aa-I domain. Specific constructs include: i) sequences from exon 5 (amino acids 127-353 of SEQ ID NO: 2) preceding the pending construct, ii) the EF site repeats (amino acids 17-603 of SEQ ID NO: 2) following the I domain, and iii) the α-chain truncated at the transmembrane region (amino acids 17-1029 of SEQ ID NO: 2) with an IgG4 tail for purification and detection purposes. These constructs are ligated into either the mammalian expression vector pDCS1 or the prokaryotic expression vector pGEX-4T-3 (Pharmacia) and the sequenced I domain. The fusion proteins are then expressed in COS, CHO or E. coli cells transformed or transfected with an appropriate expression construct. Protein is purified on a ProSepA column (Bioprocessing Limited, Durham, England), tested for reactivity with the anti-IgG4 monoclonal antibody HP6023 and visualized on polyacrylamide gels with Coomassie staining.

To construct an expression plasmid for the entire Od polypeptide, pATM.D12, described above, is modified to express an Od-IgG4 fusion protein using the following method. DNA encoding IgG4 is isolated from the vector pDCS1 by PCR using primers that individually incorporate a 5<1->AatII restriction site (SEQ ID NO: 89) and a 31-XJbaI restriction site (SEQ ID NO: . NO. 90).

Plasmid pATM.D12 is digested with Aati I and XbaI, and the appropriately digested and purified IgG4 PCR product is ligated into the linear vector.

Example 15

Production of human Qd-specific antibodies

A. Production of monoclonal antibodies

1. Transiently transfected cells from Example 7 were washed three times in Dulbecco's phosphate buffered saline (D-PBS) and injected at 5 x 10<6> cells/mouse into Balb/c mice with 50 µg/mouse muramyl dipeptidase (Sigma) in PBS. Mice were injected two or more times in the same manner at 2-week intervals. The pre-stopped and immunized serum from the mice was Bscreened by FACS analysis, as outlined in Example 9, and the spleen from the mouse with the highest reactivity to cells transfected with Od/CD18 was pooled. Hybridoma culture supernatants were then screened separately for lack of reactivity against COS cells transfected with CD11a/CD18 and for reactivity with cells cotransfected with an Od expression plasmid and CD18.

This method did not provide any monoclonal antibodies. 2. As an alternative to the production of monoclonal antibodies, soluble Od-I domain/IgG4 fusion protein was affinity purified from the supernatant of stably transfected CHO cells and used to immunize Balb/c mice, as described above. Hybridomas were established, and the supernatant from these hybridomas was screened by ELISA for reactivity against dd-I domain fusion protein. Positive cultures were then analyzed for reactivity with untruncated Od/CD18 complexes expressed on CHO transfectants.

Mice 1908 received three initial immunizations with ad/CD18 transfected CHO cells and two additional booster immunizations with soluble ad/CD18 heterodimer. Two final immunizations included 50 µg/mouse Od-I domain/IgG4 fusion protein. The fusion produced 270 IgG-producing wells. Supernatant from 45 wells showed at least 7 times higher binding to Iad/IgG4 fusion protein than to human IgG4, shown by ELISA. None of the supernatants reacted with aa/CD18-transfected CHO cells, as determined by FACS analysis.

To determine whether the supernatants were able to recognize integrin α-subunit proteins in a different context, fresh frozen spleen sections were stained with supernatants from 24 of the 45 wells. Three supernatants were determined as positive: one stained large cells in the red pulp, while two others stained scattered cells in the red pulp and also trabeculae.

These supernatants were further analyzed by their ability to immunoprecipitate biotinylated CD18 complexes from either Od/CD18-transfected CHO cells or PMA-stimulated HL60 cells. Fusion wells with supernatants that recognized protein in detergent lysates (which should not be as structurally constrained as protein expressed as heterodimers) were selected for further subcloning. Monoclonal antibodies that recognize protein in detergent may be more useful in immunoprecipitation of heterodimeric complexes from transfectants, tissues and cell lines.

3. As another alternative to monoclonal antibody production, CD18 complexes were immunoprecipitated from human spleen lysates with the anti-CD18 monoclonal antibody 23F2G after preclearing CD11a/CD18 (using monoclonal antibody TS2/4) and CD11b/CD18 (by use of monoclonal antibody Mo-1). Five Balb/c mice, 10-12 weeks old, were immunized by subcutaneous injection with approx. 30 µg of the resulting protein in complete Freund's adjuvant on day 0, followed by two booster injections of 30 µg immunogen/mouse on days 28 and 43 in incomplete Freund's adjuvant. Test sera were drawn 10 days after the last booster injection, and reactivity was determined using a 1:500 dilution of each serum to detect 1 pg/well of immunogen in a Western blot. In sera from three mice, a band of approx. 95 and 150 kD; no signal was observed in fields treated with 1:50 dilution of preimmune sera. The 150 kD band was thought to represent dd in an in vivo glycosylation state. Everyone

postimmune sera also immunoprecipitated protein from lysates from biotinylated Od/CD18-CHO cells that migrated at approximate molecular weights on SDS-PAGE, representing the heterodimer. From these results, mice No. 2212 were selected and were further immunized by intraperitoneal injection on day 64 with 30 pg of immunogen in PBS. The mouse was sacrificed 4 days later, and the spleen was sterilely removed. A single cell suspension was formed by rubbing the spleen between the ground ends of two microscope slides immersed in serum-free RPMI 1640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin and 100 pg/ml streptomycin (RPMI) (Gibco, Canada ). The cell suspension was filtered through a sterile 70-mesh Nitex cell filter (Becton Dickinson, Parsippany, New Jersey), and the filtrate was washed twice by centrifugation at 200 x g for 5 min. The resulting pellet was resuspended in 20 ml of serum-free RPMI. Thymocytes taken from three natural Balb/c mice were prepared in a similar manner.

Before fusion, NS-1 myeloma cells maintained in log phase in RPMI with 10% Fetalclone serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah) for 3 days before fusion were pelleted by centrifugation at 200 x g for 5 min, washed twice as described in the preceding paragraph and the count. Approximately 2 x 10<8> spleen cells were combined with 4 x 10<7> NS-1 cells, and the resulting mixture was pelleted by centrifugation at 200 x g. The supernatant was discarded. The cell pellet was dislodged by tapping the tube, and 2 ml of 50% PEG 1500 in 75 mM HEPES

(pH 8.0, 37 °C) (Boehringer Mannheim) was added during

1 minute while stirring. An additional 14 ml of serum-free RPMI was then added over the next 7 min, followed immediately by the addition of 16 ml of RPMI. The resulting mixture was centrifuged at 200 x g for 10 min, and the supernatant

was thrown. The pellet was resuspended in 200 ml RPMI containing 15% FBS, 100 mM sodium hypoxanthine, 0.4 mM aminopterin, 16 mM thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim) and 1.5 x IO< 6> thymocytes/ml, and transferred to ten 96-well, flat-bottom tissue culture plates (Corning, UK) in an amount of 200 pg/well. Cells were supplied with nutrients

days 2, 4 and 6 after fusion by aspiration of approx. 100 µl from each well with an 18 G needle (Becton Dickinson) and addition of 100 µl/well diffusion medium, described above, except that it contained 10 units/ml IL-6 and lacked thymocytes.

On days 7-10 post-fusion, supernatant from each well was screened by antibody capture ELISA as a test for the presence of mouse IgG. Immulon 4 plates (Dynatech, Cambridge, Massachusetts) were coated with 50 µl/well goat anti-mouse IgA, -IgG, or -IgM (Organon Teknika) diluted 1:5000 in 50 mM carbonate buffer, pH 9.6, at 4° C. Plates were washed three times with PBS containing 0.5% Tween 20 (PBST), and 50 µl of culture supernatant from each well was added. After incubation at 37°C for 30 min, wells were washed with PBST, as described above, and 50 μl of horseradish peroxidase-conjugated goat anti-mouse IgG(fc) (Jackson Immuno-Research, West Grove, Pennsylvania) diluted 1:3500 in PBST was added to each well. The plates were incubated as described above, washed four times with PBST and added 100 µl substrate consisting of 1 mg/ml o-phenylenediamine (Sigma) and 0.1 µl/ml 30% H 2 O 2 in 100 mM citrate, pH 4.5. The color reaction was stopped after 5 minutes by the addition of 50 µl of 15% H 2 SO 4 . Absorbance at 490 nm was determined for each well using a plate reader (Dynatech).

Hybridomas were further characterized as follows. Supernatants from IgG-producing cultures were analyzed by flow cytometry for reactivity to α<i/CD18-transformed CHO cells but not to JY cells (a B cell line positive for LFA-1 but not for other β2 integrins , as observed in previous staining experiments in the laboratory). Briefly, 5 x 10<5>Od/CD18-transformed CHO or Od/CD18"JY cells were suspended in 50 µl RPMI containing 2% FBS and 10 mM NaN3 (FACS buffer). Individual cell suspensions were added to 50 µl igG-positive hybridoma culture supernatant in wells of 96-well, round-bottom plates (Corning). After incubation for 30 min on ice, cells were washed twice by pelleting in a clinical centrifuge, supernatant from each well was discarded, and pellets were resuspended in 200- 300 µl FACS buffer The last wash was replaced with 50 µl/well of a 1:100 dilution of an F (ab")2 fragment of sheep anti-mouse IgG(H + L)-FlTC conjugate {Sigma , St. Louis, Missouri) prepared in FACS buffer. After incubation, as described above, cells were washed twice with Dulbecco's PBS {D-PBS) supplemented with 10 mM NaN 3 , and finally resuspended in D-PBS containing 1% paraformaldehyde. Samples were then transferred to polystyrene tubes for flow cytometric analysis (FACS) with a Becton Dickinson FACScan analyzer.

The fusion produced four cultures that were judged positive by both criteria. When the second Bcreening was repeated on expanded supernatants approx. 4 days later, three of the four cultures remained positive. Three wells, designated 169A, 169B and 169D, were then successively cloned 2-3 times by 2-fold dilution in RPMI, 15% FBS, 100 mM sodium hypoxanthine, 16 mM thymidine and 10 units/ml IL-6. Wells on clone plates were counted visually after 4 days; and the number of colonies in the least dense wells was recorded. Selected wells from each cloning were analyzed by FACS after 7-10 days. Activity was found in two of the cultures, 169A and 169B. In the final cloning, positive wells containing single colonies were expanded in RPMI with 11% FBS. Antibody from clonal supernatants of 169A and 169B was isotyped using the IsoStrip kit (Boehringer Mannheim) according to the manufacturer's instructions and found to be of the IgG1 isotype.

Immunoprecipitation of Od/CD18 complexes from CHO transfectants and PMA-stimulated HL60 cells was used as a tertiary screen for specificity. Hybridomas 169A and 169B precipitated appropriate bands from CHO lines and a single α-chain type of 150-160 kD from HL60 cells, as determined by SDS-PAGE. Hybridomas 169A and 169B were deposited on May 31, 1995 at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, and given accession numbers HB11907 and HB11906, respectively.

To more fully characterize the binding properties of 169A and 169B, the ability of each antibody to inhibit binding of the other antibody or anti-CD18 antibody TS1/18.1 to soluble o.d/CD18 was tested. Soluble, untruncated Od/CD18 was immobilized separately using an unlabeled antibody in a 96-well plate format, and biotinylated antibodies were used to detect protein bound by the same, or other, unlabeled antibodies. Binding was detected using a goat antimouse Ig/HRP conjugate, followed by the addition of OPD substrate. The results indicated that antibody 169A was able to block binding of biotinylated 169A and TS1/18.1, while antibody 169B only blocked binding of itself. 4. Another mouse (no. 2214), immunized using the same protocol as mouse no. 2212, was selected and further immunized using a pre-fusion boost injection on day 70 with 30 µg of purified eid from spleen lysates in PBS. The mouse was sacrificed 4 days later, and the spleen was sterilely removed.

The fusion and cloning of positive cells were performed as described above. The fusion produced five anti-Od monoclonal hybridomas designated 17OD, 17OF, 17OE, 170X, and 170H, which were isotyped as IgGi using the IsoStrip kit (Boehringer Mannheim) in accordance with the manufacturer's instructions. 5. Yet another mouse, #2211, immunized using the same starting protocol as mouse #2212 and mouse #2214, was selected and further immunized on day 88 with 30 µg of immunogen and a pre-fusion boost injection of 30 µg of immunogen on day 2 03. The mouse was sacrificed 4 days later, and the spleen was removed and the fusion performed as described above. Hybridoma supernatant was screened by antibody capture ELISA and by flow cytometry, as detailed in the sections above.

Fifteen positive hybridomas were identified, designated 188B, 188C, 188E, 188F, 188G, 1881, 188J, 188K, 188L, 188M, 188N, 188P, 188R AND 188T, and wasotyped in ELISA analysis. Briefly, Immulon 4 plates (Dynatech, Cambridge, Massachusetts) were coated at 4 °C with 50 µl/well goat anti-mouse IgA, -G, -M (Organon Teknika) diluted 1:5000 in 50 mM carbonate buffer, pH 9 ,6. Plates were blocked in

30 min at 37°C with 1% BSA in PBS, washed three times with PBS/0.05% Tween 20 (PBST), and 50 µl of culture supernatant (diluted 1:10 in PBST) was added. After incubation and washing, as described above, 50 µl of horseradish peroxidase-conjugated rabbit anti-mouse IgGi, -G2a, or -G3 (Zymed, San Francisco, Calif.) diluted 1:1000 in PBST with 1% normal goat serum was added. The plates were incubated as described above, washed four times with PBST and then added with 100 µl substrate consisting of 1 mg/ml o-phenylenediamine (Sigma) and 0.1 µl/ml 30% H 2 O 2 in 100 mM citrate, pH 4.5. The color reaction was stopped after 5 minutes by the addition of 50 µl of 15% H 2 SO 4 . A490 was read on a plate reader (Dynatech) and all 15 antibodies were determined to be IgG1. The surplus of spleen cells from mouse no. 2211 was frozen in a cryopoule and stored in liquid nitrogen. The cryopoule was rapidly thawed by placing it in a 37°C water bath and rotating it until the contents were melted. The cells were transferred to a 15 ml centrifuge tube, and warm RPMI containing 11% FBS was added slowly in portions of 1 ml at 3-5 minute intervals. An additional 5 ml of warm RPMI was added and after 5 min the tube was centrifuged at 200 x g for 5 min and the supernatant was aspirated. Cells were resuspended in RPMI, and a fusion was performed as described above. Hybridoma supernatant was screened by antibody capture and flow cytometry, as described above.

The fusion produced five clones designated 195A, 195C, 195D, 195E and 195H. The clones were isotyped using the ELI SA procedure, as described above; monoclonal antibodies 195A, 195C, 195D and 195E were determined to be IgG1f and 195H was determined to be IgG2a.

6. To identify antibodies capable of inhibiting functional aa binding, soluble aa/CD18LZ (see Example 14) is used for immunization. The protein is isolated on an affinity chromatography resin from supernatant from short-term transfected COS cells, and the resin-bound Oa is used as an immunogen. A selected mouse is immunized as described above and given a final booster injection 2 weeks after the original immunization. Immunization using this technique prevents possible changes in protein conformation, which are often associated with detergent lysis of cells. Additional mice are immunized with recombinant protein, also resin-bound, but were not originally immunized with protein purified from cell lysate.

Hybridomas, prepared as described above, resulting from the immunization are screened by ELISA of the recombinant protein immobilized from a cell supernatant using the Fab fragment of a non-blocking antibody. Alternatively, flow cytometry is used to analyze for reactivity towards JY cells that have previously been transfected with ctd cDNA. 7. Alternatively, monoclonal antibodies are generated as follows. Affinity-purified a,j/CD18-heterodimer protein from detergent lysates from stably transfected CHO cells is used with 50 ug/ml muramyl peptidase to immunize Balb/c mice, as described above. Mice receive three immunizations before serum reactivity against Od/CD18 is determined by immunoprecipitation of biotinylated complexes in the CHO transfectants. Hybridomas from positive animals are established according to standard protocols, after which hybridoma cultures are selected by flow cytometry using cid/CD18 transfectants. CD11a/CD18 transfectants are used as a control for CD18 reactivity only. 8. As another alternative for producing monoclonal antibodies, Balb/c mice undergo an immunization/suppression protocol designed to reduce reactivity to CHO cell determinants on transfectants used for immunization. This protocol involves immunization with untransfected CHO cells and subsequent killing of CHO-reactive B-cell blasts by cyclophosphamide treatment. After three rounds of immunization and cyclophosphamide treatment have been carried out, the mice are immunized with ctd/CD18-CHO-transfected cells, as described above. 9. Alternatively, complexes are enriched from detergent lysates of PMA-stimulated HL60 cells by pre-clearing, as described above. Other pVintegrins are clarified on the same columns. Immunization with the resulting complexes, hybridoma production and screening protocols are performed as described above.

B. Production of polyclonal sera

Purified Od-I domain/IgG4 chimeras (Example 14) were used to generate polyclonal antiserum in rabbits. The Od-I domain/IgG4 antigen was first injected in an amount of

100 pg/rabbit in complete Freund's adjuvant, followed by three booster injections with the same amount of protein in incomplete Freund's adjuvant. Blood samples were analyzed after the third and fourth injections. Rabbit immunoglobulin (Ig) was purified from serum on a protein A-Sepharose column and precleared for antihuman IgG reactivity on a human IgG/Affigel 10 column. Reactivity by ELISA to the I-domain chimera, but not to human IgG, was used to confirm complete preclearance.

The pre-cleared polyclonal sera were used to immunoprecipitate protein from detergent lysates from surface biotinylated CHO cells previously transfected with Od and CD18 expression vectors. Immunoprecipitation was performed using the previously described method in Example 10. The pre-cleared sera recognized a protein complex of the same molecular weight as that precipitated by anti-CD18 monoclonal antibody TS1.18. The sera also recognized a single band of appropriate size in a Western blot of CD18 complexes from ad/CD18-transfected CHO cells. Affinity-purified integrins CDlla/CD18, CDllb/CD18 and VLA4 from human spleen were not recognized by the rabbit polyclonal sera. The sera did not react with Od-transfected CHO cells in solution, as determined by flow cytometry. It was therefore concluded that the polyclonal rabbit sera were only able to recognize denatured ad-I domain/IgG4 proteins.

In an attempt to produce polyclonal antisera against ad/CD18, a mouse was immunized three times with ad-transfected CHO cells (D6.CHO, Od/CD18) with adjuvant peptide and once with purified dd/CD18 heterodimer. A final boost injection included only ad/CD18 heterodimers. Approximately 100 ul of immunized serum was pre-clarified by adding approx. 10s LFA-1-transfected CHO cells for 2 h at 4 °C. The resulting serum was assayed for eID reactivity at dilutions of 1/5000, 1/10000, 1/20000 and 1/40000 on normal human spleen. The polyclonal antibody was reactive at a dilution of 1/20,000, while a 1/40,000 dilution stained very weakly.

Example 16

Analysis of ag distribution

Tissue distribution of Od/CD18 was determined using polyclonal antiserum generated as described in Example 15.

Purified rabbit polyclonal antibody was used at concentrations ranging between 120 ng/ml and 60 pg/ml for immunocytochemical analysis of frozen human spleen sections. Sections of 6 micron thickness were placed on Superfrost Plus slides (VWR) and stored at -70 °C. Before use, the slides were removed from -70 °C and placed at 55 °C for 5 minutes. The sections were then fixed in cold acetone for 2 minutes and air-dried. The sections were blocked in a solution containing 1% BSA, 30% normal human serum and 5% normal rabbit serum for 30 minutes at room temperature. Primary antibody was applied to each section for 1 hour at room temperature. Unbound antibody was removed by washing the slides three times in TBS buffer for 5 minutes each. wash. A rabbit anti-mouse IgG binding antibody was then applied to each section in the same TBS buffer. A mouse alkaline-phosphatase-anti-alkaline-phosphatase (APAAP) antibody, incubated for 30 minutes at room temperature, was used to detect the second antibody. The slides were then washed three times in TBS buffer. Fast Blue substrate (Vector Labs) was applied, and color development was stopped by immersion in water. The slides were counterstained in Nuclear Fast Red (Sigma) and rinsed in water before mounting with Aqua Mount (Baxter). Staining was detected in the red splenic mass with a reagent, but not with an irrelevant rabbit polyclonal Ig preparation or the crude preimmune serum from the same animal.

A mouse serum was determined to have specific ctd reactivity and this was used to stain various lymphoid and non-lymphoid tissues. Monoclonal antibodies recognizing CD18, CDlla, CDllb and CDllc were used in the same experiment as controls. Staining of normal spleen sections with ad-polyclonal sera and monoclonal antibodies against CDlla, CDllb, CDllc and CD18 revealed the following results. The pattern observed with cid polyclonal sera did not exhibit the same labeling pattern as CDlla, CDllb, CDllc or CD18. There is a distinct labeling pattern with some cells located in the marginal zone of the white matter, and a distinct labeling of cells peripheral to the marginal zone. This pattern was not observed with the other antibodies. Individual cells scattered throughout the red pulp were also labeled, and these cells may or may not be the same population or subset observed with CDlla and CD18.

Labeling with CDllc showed some cells staining in the marginal zone, but the antibody did not show the distinct ring pattern around the white mass compared to Od polyclonal sera, nor did labeling in the red mass give the same staining pattern as Od polyclonal sera.

The labeling pattern observed with Od polyclonal serum was therefore unique compared to the pattern observed using antibodies against the other β2 integrins (CDlla, CDllb, CDllc and CD18) and suggests that the in vivo distribution of dd in humans is different from that of other p2 -integrins.

Characterization of human Od expression with monoclonal antibodies

Antibodies secreted by hybridomas 169A and 169B were used to analyze human Ad expression in frozen tissue sections by immunocytochemistry, and on cell lines and peripheral blood leukocytes by flow cytometry. Hybridoma supernatants used in both sets of experiments were undiluted.

Tissue staining

All stainings were performed as described above, with the exception of liver sections, which were stained as follows. After acetone fixation, sections were cured in 1% H 2 O 2 and 1% sodium azide in TBS for 15 min at room temperature. After primary antibody staining, a rabbit anti-mouse antibody, directly conjugated with peroxidase, was applied for 30 minutes at room temperature. Slides were washed three times in TBS buffer. A pig anti-rabbit antibody, directly conjugated to peroxidase, was incubated for 30 minutes at room temperature to detect the second antibody. Slides were then washed three times in TBS buffer, and AEC substrate (Vector Labs) was applied to allow color development. Slides were counterstained with hematoxylin, Gill's No. 2 (Sigma), and then rinsed in water before dehydration and mounting.

In spleen sections, the majority of expression was localized to the red splenic mass in cells identified by morphology as granulocytes and macrophages. A large number of granulocytes were stained, while only a subset of macrophages gave signal. A small number of follicular dendritic cells in the white matter were also weakly stained by the aa antibodies. CDlla and CD18 staining was detected throughout the red and white pulp. CDllc staining was more pronounced in large cells, which were thought to be macrophages in the white splenic mass and in the marginal zone surrounding the white pulp, diffuse staining in the red pulp was also noted. CDllb appeared to have a distribution that overlapped, but was not identical to, Od in the red pulp without any white pulp involvement.

Integrin expression in normal and (rheumatoid) arthritic synovial tissue was compared. Minimal staining with all anti-integrin antibodies (including antibodies specifically immunoreactive with CD11a, CD11b, CD11c, CD18, as well as aa) was observed in normal tissue with a wide distribution on local cells, presumably macrophages. In the inflamed synovium, expression of all integrins was more localized to cells clustered around lymphatic vessels. Although the dd and CD11b expression patterns were similar, CD11c did not appear to be as highly expressed and was restricted to a subset of leukocytes.

In dogs, expression of CD11b, but not dd, was observed on liver macrophages or Kuppfer cells. Staining of normal human liver sections (as previously described for staining of dog liver sections, supra) confirmed conservation of this staining pattern in humans. CDllc was also detected at low levels. In a section from a hepatitis patient, all leuko-integrin staining was higher than that observed on normal liver, while expression was detected on macrophages and granulocytes in these samples.

Minimal staining of normal human colon sections was observed with anti-Ad antibody; weak staining of smooth muscle and leukocytes was observed. All leukointegrins were detected at higher levels on average from patients with Crohn's disease.

Normal lung showed a limited number of weakly Ad-positive cells; these were determined by morphology to be macrophages and neutrophils. In lung tissue from a patient with emphysema, dd staining was observed on neutrophils and macrophages containing hemosiderin, an iron-containing pigment, indicating that these cells have engulfed red blood cells.

Sections from normal brain and plaque lesions from patients with multiple sclerosis (MS) were examined for integrin expression. In normal brain our c^-staining less intense than with CDlla, CDllb and CDllc and limited to cells typed as microglial cells by morphology and CD68 staining. CD11b-positive cells were located around blood vessels and throughout the tissue. CDllc<+-> cells appear to be localized in blood vessels, while ein cells surround the blood vessels. In MS tissue sections, c^ expression was found on both microglial cells and on a non-macrophage leukocyte subset; ad<+->cells were located in plaque lesions and throughout the cortex. The aa signal had an intensity equivalent to CDllc but lower than CDllb.

Both thoracic aortic and abdominal aortic sections from PDAY (Pathobiological Determinants of Atherosclerosis in Youth, LSU Medical Center) tissue samples were analyzed with antileukointe-grin and anti-CAM antibodies. The lesions examined were consistent with aortic fatty streaks consisting of subintimal aggregates of large foam cells (mainly macrophages with digested lipid) and infiltrates of smaller leukocytes. Single-marker studies with monoclonal antibodies specific for Cm and the other β2-integrin α chains (CDlla, CDllb and CDllc) plus a macrophage marker (CD68) revealed that the majority of lipid-laden macrophages expressed a moderate level of 0$ and CD18, whereas CDlla and CDllc were expressed at weak or weak to moderate levels, respectively. CD11b was weakly expressed and then only by a subset of macrophages.

Dual marker studies were performed to determine it

relative localization of ad and ICAM-R antigens in aortic sections. Because foam cells in these sections were stained with the antibody Ham 56, which is specific for a macrophage marker, but not with antibodies to smooth muscle actin, it was determined that the foam cells were not derived from subintimal smooth muscle cells. CD68-positive macrophages expressing aa were surrounded and interspersed with small ICAM-R-positive leukocytes. There appeared to be a limited number of small leukocytes that were CD68 negative but stained with both Od and ICAM-R antibodies.

Distribution of Od in normal tissues appears to be localized to leukocytes in a pattern that overlaps, but is not identical to, that of CDllb and CDllc, two other leuko-integrin-α chains that have previously been characterized as having restricted leukocyte distribution . Cellular morphology indicated that Ad staining is largely restricted to macrophages and granulocytes, with limited lymphocyte staining. Tissue inflammation generally appeared to increase the number and types of leukocytes observed in a particular tissue, along with increased staining of leukointegrins, including ad- Because the cellular and spatial distribution of leukointegrins was not identical in pathological tissues, it was concluded that distinct functions and ligands exist for each family member, including Od, in specific contexts.

It is interesting that Od expression in early atherosclerotic lesions appeared to be more pronounced than the expression of CDlla, CDllb and CDllc, suggesting that Od may play a central role in the establishment of these lesions. The distribution of ad- and ICAM-R-positive cells, supported by evidence suggesting an interaction between aa and ICAM-R, suggests that Od may be involved in leukocyte recruitment or activation at early stages in these lesions.

Cell line and peripheral blood leukocyte staining

Antibodies 169A and 169B stained a promyelomonocytic cell line, HL60, by FACS. Surface expression of eid in these cells is negatively affected by PMA stimulation, which is reported to induce differentiation along a macrophage pathway, but is unaffected by DMSO which induces granulocyte differentiation [Collins et al., Blood, 70:1233-1244 (1987)]. The FACS profiles for 169A and 169B with PMA stimulation were antithetical to those observed with anti-CD11b and anti-CD11c monoclonal antibodies. A monocyte cell line, THP-1, also showed weak staining with 169A and 169B. A subset of cells in the lymphocyte and monocyte inlets of peripheral blood leukocytes also appeared to be weakly positive by FACS. A subset of peripheral blood monocytes stained weakly with 169A and 169B, while B lymphocytes were found to have no surface expression of Od- CD8<+->the subset of T lymphocytes was dd<+.> Moreover, antibodies 169A and 169B did not recognize antigen on B-cell lines JY, Ramos, a basophilic line, KU812, and T-cell lines Jurkat, SKW and Molt 16.

In light of the results with HL60 cells, granulocytes were isolated from peripheral blood by Ficoll/Hypaque gradient centrifugation and subsequent lysis of red blood cells. All preparations were found to consist of > 90% PMN by visualization of nuclear morphology in acetic acid. Separate populations were stimulated for 30 minutes with 50 ng/ml PMA or 10"e M formyl peptide (fMLP) to release potential intracellular integrin stores. Unstimulated populations showed low, but significant, expression of 169A and 169B antigens relative to an IgG1 -control, with a detectable increase observed upon stimulation. On PMN, the level of eid and CDllc surface expression was more similar to that observed on HL60 cells. The antibody 169B was then used to precipitate a heterodimer molecule from a detergent lysate of biotinylated PMN with subunit sizes of approximately 150 and 95 kD, which respectively fit Od and CD18.

The presence of Ad on PMN could not be expected from the known information on dog Ad expression. Canine neutrophils, unlike their human counterparts, express the T helper cell marker CD4 and also the integrin VLA-4, and therefore may have different ligands and functions in dogs than in

human.

Coloring of PBL subgroups

The present study was conducted to determine the distribution of this β2 integrin in human peripheral blood leukocytes. The cell surface density of dd in relation to other β2 integrins was also compared. The acute regulation of eid expression in purified human eosinophils was finally also evaluated.

Human peripheral blood leukocytes were separated by density gradient centrifugation into a mononuclear cell fraction (containing monocytes, lymphocytes and basophils)' and granulocytes (neutrophils and eosinophils) [Warner et al., J. Immunol. Meth., 105:107-110 (1987)]. For some experiments, eosinophils were purified using CD16 immunomagnetic selection to purities higher than 95% [Hansel et al., J. Immunol. Meth., 122:97-103 (1989)]. Skin mast cells were enzymatically dispersed from human skin and enriched as described previously [Lawrence et al., J. Immunol., 139:3062-3069

(1987)].

Cells were labeled with appropriate dilutions of monoclonal antibody specific for either CDlla (MHM24), CDllb

(H5A4), CDllc (BU-15) or dd (169A). A mouse control IgGi was also used. Cells were washed and then incubated with phyco-erythrin conjugated goat anti-mouse IgG. In some experiments, cells were incubated with an excess of mouse IgG and FITC-labeled mouse monoclonal antibody or goat polyclonal antibody specific for a particular cell (eg, CD3, CD4, or CD8 for T cells; CD16+ lymphocytes for NK cells; anti-IgE for basophils [Bochner et al., J. Immunol. Meth., 125:265-271 (1989)]. The samples were then examined by flow cytometry (Coulter EPICS Profile) using appropriate signal -control to identify cell subsets.

For studies with human eosinophils in which acute upregulation of Od expression was examined, cells were stimulated for 15 minutes at 37°C with phorbol ester (10 ng/ml), RANTES (100 ng/ml) [Schall, Cytokine, 3:165- 183 (1991)] or IL-5 (10 ng/ml) before labeling with the different monoclonal

Results showed that ad was present on all peripheral blood eosinophils, basophils, neutrophils, monocytes and NK cells. A small subset (about 30%) of CD8<+->lymphocytes was also found to express eid. Skin mast cells and CD4<+->lymphocytes did not express aa. CDlla and CDllb are generally present at a higher density on leukocytes than ad, the latter being expressed at relatively low levels similar to CDllc. Among leukocytes, monocytes and CD8<+-> cells have the highest density of Oa, while eosinophils have the lowest level of c^ expression. Expression on neutrophils, basophils and NK cells was in an intermediate position.

Stimulation of peripheral eosinophils with the CC chemokine RANTES caused no change in the expression of any of the β2 integrins. However, treatment with phorbol ester produced a 2-3 fold increase in expression of both CD11b and ad, but did not cause expression of CD11a or CD11c. IL-5 treatment resulted in selective upregulation of CD11b expression without affecting levels of the other integrin subunits.

Taken together, these results indicate that in peripheral blood leukocytes, cia is usually expressed at a level comparable to CD11c. The highest levels are found on monocytes and a subset of CD8<+->lymphocytes. Human skin mast cells do not express cia. Purified eosinophils appear to have intracytoplasmic stores of CD11b and aa- The differential up-regulation shown by means of IL-5 in relation to PMA, however, suggests that these stores are separate from each other.

Staining patterns for peripheral blood leukocyte (PBL) subsets were also determined by flow cytometry using a combination of signal selection and surface markers, as described above, in an attempt to more accurately define the 169A/B-negative lymphocyte population. PBL were isolated on ficoll, as described previously, and stained separately with 169A, 169B and monoclonal antibodies against CD14 (monocyte/macrophage marker), CD20 (B cell), CD56 (NK cell), T cell receptor a/ p (T cell), CD16 (neutrophils, NK) and a4 (a negative marker for neutrophils). Outlets were defined using size and marker distribution.

Results indicated that cells in the CD14+ monocyte efflux exhibited low levels of 169A and 169B staining. A bimodal expression pattern observed in previous experiments in the lymphocyte egress was resolved by increasing forward scattering. The mixed TCR<+>/CD20<+-> population appears to have low, but homogeneous, levels of 169A/B expression, while a population mapped at slightly higher side scatter (cellular complexity), which gave 50% positive staining for CD56, appears to have a distinct 169A/B-negative population. The negative population was thus not recognized by TCR, CD20, CD14 or CD16 antibodies.

Synovial distribution of Qd

To determine the cellular distribution of aa, other β2-integrins and their counter-receptors in inflammatory and non-inflammatory synovium, monoclonal antibodies against the various β2-integrin and immunoglobulin supergene families were used in immunohistological investigations. Protein expression was determined in normal, osteoarthritic and rheumatoid synovial tissue samples.

Results indicated that the cell layer of the synovial membrane expressed high levels of VCAM-1, CD11b/CD18 and Od/CD18. In these cells, CD11c/CD18 expression is limited, and CD11a/CD18 is usually not detected. In rheumatoid arthritis synovitis, expression of |32-integrins in the synovial cell layer increases proportionally with the degree of hyperplasia. The proportion of cells expressing CDllc increases significantly and approaches the expression of CDllb and aa, but there is no increase of CDlla expression.

In the tissue areas below the membrane, aggregates and diffuse infiltrates of CD3/CDlla/ICAM-R<*->lymphocytes are scattered among CD68/CDllb/ad+ macrophages. A significant number of aggregates show intense cta staining, especially in T cell-rich areas.

The synovial endothelium expressed variable ICAM-1 and ICAM-2 with minimal evidence of ICAM-R expression.

Taken together, these results indicate that synovial macrophages and macrophage-like synovial cells constitutively express high levels of the β2 integrins CD11b and ctd. In synovitis, there is an expansion of this subset of cells in both the membrane and submembrane areas, together with an apparent increase in expression of CD11c. Thus, in addition to expressing CDlla and ICAM-R, specific populations of rheumatoid synovial T-lymphocytes express high levels of aa where the latter molecule above has been shown to be expressed at low levels by peripheral blood lymphocytes.

Example 17

Isolation of rat cDNA clones

In view of the existence of both canine and human dd subunits, attempts were made to isolate homologous genes in other species, including rat (this example) and mouse (Example 17 below).

A partial sequence of a rat cDNA showing homology to the human Od gene was obtained from a rat spleen Agt10 library (Clontech). The library was spread at 2 x 10<4> pfu/plate on 150 mm LBM/agar plates. The library was blotted onto Hybond membranes (Amersham), denatured for 3 minutes, neutralized for 3 minutes and washed for 5 minutes with buffers, as described in standard protocols [Sambrook et al., Molecular Cloning: A Laboratory Manual, p. 110 ]. The membranes were immediately placed in a Stratalinker (Stratagene), and the DNA was cross-linked using the automatic cross-link setting. The membranes were prehybridized and hybridized in 30% or 50% formamide, for low- and high-stringency conditions, respectively. The membranes were first screened with a <32>P-labeled probe generated from the human ctd cDNA, which corresponds to bases 500-2100 of clone 19A2 (SEQ ID NO: 1). The probe was labeled using Boehringer Mannheim's "Random Prime Kit" in accordance with the manufacturer's suggested protocol. The filters were washed twice with SSC at 55 °C.

Two clones designated 684.3 and 705.1 were identified, which showed sequence homology with human α^, human CD11b and human CD11c. Both clones matched the human ctd gene in the 3<1> region of the gene, from base 1871 to base 3012 for clone 684.3 and bases 1551-3367 for clone 705.1.

To isolate a more complete rat sequence that included the 5' region, the same library was screened again using the same protocol used for the initial screen, but using a mouse probe generated from clone A1160 (see Example 17 below) . Single isolated plaques were selected from the second screening and maintained as single clones on LBM/agar plates. Sequencing primers 434FL and 434FR (SEQ ID NO: 34 and 35, respectively) were used in a standard PCR protocol to generate DNA for sequencing.

DNA from PCR was purified using a Quick Spin column (Qiagen) in accordance with the manufacturer's suggested protocol.

Two clones designated 741.4 and 741.11 were identified, overlapping clones 684.3 and 705.1; in the overlapping regions, clones 741.1 and 741.11 were 100% homologous to clones 684.3 and 705.1. A composite rat cDNA with homology to the human ctd gene is shown in SEQ ID NO. NO. 36; the calculated amino acid sequence is shown in SEQ ID NO. NO. 37.

Cloning of the 5' end of rat aj

A 5* cDNA fragment of the rat aa gene was obtained using a Clonetech rat spleen RACE cloning kit in accordance with the manufacturer's suggested protocol. The gene-specific oligonucleotides used were designated 741.11#2R and 741.2#1R (SEQ ID NO: 59 and 58, respectively).

Oligo 74l.ll#2R comprises base pairs 131-152 in SEQ ID NO. NO. 36, in the opposite orientation, and 741.2#1R comprises base pairs 696-715 in SEQ ID NO. NO. 36, also in the opposite orientation. A primary PCR was performed using the most 3' oligo, 741.2#1R. A second PCR was performed using oligo 741.11#2R and DNA generated from the primary reaction. A band of approx. 300 base pairs were detected on a 1% agarose gel.

The second PCR product was ligated into plasmid pCRTAII (Invitrogen) in accordance with the manufacturer's suggested protocol. White (positive) colonies were picked and added to 100 µl of LBM containing 1 µl of a 50 mg/ml carbenicillin stock solution and 1 µl of M13 K07 phage culture, in individual wells of a round-bottom, 96-well culture plate. The mixture was incubated at 37°C for 30 minutes to 1 hour. After the initial incubation period, 100 µl of LBM (containing 1 µl of 50 mg/ml carbenicillin and a 1:250 dilution of a 10 mg/ml kanamycin stock solution) was added and incubation was continued overnight at 37°C.

Using a sterile 96-well metal transfer fork, the supernatant from the 96-well plate was transferred to four Amersham Hybond nylon filters. The filters were denatured, neutralized and cross-linked using standard protocols. The filters were prehybridized in 20 ml prehybridization buffer (5 x SSPE; 5 x Denhardt's; 1% SDS; 50 pg/ml denatured salmon sperm DNA) at 50 °C for several hours with agitation.

Oligoprobes 741.11#1 and 741.11#1R (SEQ ID NO: 56 and 57, respectively), which comprised base pairs 86-105 (SEQ ID NO: 36) in the forward and reverse orientations, respectively, were labeled as follows.

Approximately 65 ng oligo DNA in 12 µl dH2O was heated to 65 °C in

2 minutes. 3 µl of 10 mCi/ml Y-<32p_>ATP was added to the tube along with 4 µl of 5x kinase buffer (Gibco) and 1 µl of T4-DNA kinase (Gibco). The mixture was incubated at 37°C for 30 minutes. After incubation, 16 µl of each labeled oligoprobe was added to the prehybridization buffer and filters, and hybridization was continued overnight at 42°C. The filters were washed three times in 5 x SSPE; 0.1% SDS, for 5 minutes per wash at room temperature, and autoradiograph for 6 hours. Positive clones were expanded, and DNA was purified using the Magic Mini Prep Kit (Promega) in accordance with the manufacturer's suggested protocol. Clone 2F7 was selected for sequencing and showed 100% homology to clone 741.11 in the overlapping region. The complete rat Oa nucleic acid sequence is shown in SEQ ID NO. NO. 54; the amino acid sequence is shown in SEQ ID NO. NO. 55.

Characteristic features of the rat cDNA and amino acid sequences

Neither nucleic acid sequences nor amino acid sequences have previously been reported for rat α-subunits in β2 integrins. However, sequence comparisons with reported human β2 integrin α subunits suggested that the isolated rat clone and its predicted amino acid sequence are very closely related to Od nucleotide and amino acid sequences.

At the nucleic acid level, the isolated rat cDNA clone shows 80% identity compared to the human cid cDNA; 68% identity compared to human CDllb; 70% identity compared to human CDllc; and 65% identity compared to mouse CD11b. No significant identity is found when comparing human CDlla and mouse CDlla.

At the amino acid level, the predicted rat polypeptide encoded by the isolated cDNA shows 70% identity compared to human Od polypeptide; 28% identity compared to human CDlla; 58% identity compared to human CDllb; 61% identity compared to human CDllc; 28% identity compared to mouse CDlla; and 55% identity compared to mouse CD11b.

Example 18

Production and characterization of rodent-Od-specific antibodies

A. Antibody f is against rat Ad-I domain/HuIgG4 fusion proteins

Considering the fact that the I domain of human β2 integrins has been shown to participate in ligand binding, it was hypothesized that the same would be the case for rat oa protein. Monoclonal antibodies immunospecific for the rat ctd-1 domain may therefore be applicable in rat models of human disease states in which cid binding is implicated.

Oligonucleotides "rat-α-DI5" (SEQ ID NO: 87) and "rat-α-DI3" (SEQ ID NO: 88) were generated from the rat ctd sequence and corresponded respectively to base pairs 469-4 93 and base pairs 1101-1125 (in the opposite orientation) in SEQ ID NO. NO. 54. The oligonucleotides were used in a standard PCR reaction to generate a rat Od DNA fragment containing the I domain spanning base pairs 459-1125 of SEQ ID NO. NO. 54. The PCR product was ligated into vector pCRTAII (Invitrogen) in accordance with the manufacturer's suggested protocol. A positive colony was selected and expanded for DNA purification using a Qiagen-Midi Prep kit (Chatswoth, GA) according to the manufacturer's protocol. DNA was digested with XhoI and BgrIII in a standard restriction enzyme digestion, and a band of 600 base pairs was gel purified and then ligated into pDCS1/HuIgG4 expression vector. A positive colony was selected, expanded and DNA purified with a Qiagen Maxi Prep kit.

COS cells were spread at half confluence on 100 mm culture dishes and grown overnight at 37 °C in 7% CO 2 . Cells were rinsed once with 5 ml of DMEM. To 5 ml DMEM was added 50 µl DEAE dextran, 2 µl chloroquine and 15 µg rat Od-I domain/HuIgG4 DNA, described above. The mixture was added to the COS cells and incubated at 37°C for 3 hours. The medium was then removed, and 5 mL of 10% DMSO in CMF-PBS was added for exactly 1 minute. The cells were gently rinsed once with DMEM. 10 ml of DMEM containing 10% FBS was added to the cells and incubation was continued overnight at 37°C in 7% CO 2 . The next day, the medium was replaced with fresh medium, and the incubation was continued for 3 additional days. The medium was harvested, and fresh medium was added to the plate. After 3 days, the medium was collected again and the plates were discarded. The procedure was repeated until 2 1 culture supernatant had been collected.

Supernatant collected as described above was applied to a Prosep-A column (Bioprocessing Limited) and the protein was purified as described below.

The column was first washed with 15 column volumes of wash buffer containing 35 mM Tris and 150 mM NaCl, pH 7.5. Supernatant was applied at a slow rate of less than approx. 60 column volumes per hour. After application, the column was washed with 15 column volumes of wash buffer, 15 column volumes of 0.55 M diethanolamine, pH 8.5, and 15 column volumes of 50 mM citric acid, pH 5.0. Protein was eluted with 50 mM citric acid, pH 3.0. The protein was neutralized with 1.0 M Tris, pH 8.0, and dialyzed in sterile PBS.

The rat ad I domain protein was analyzed as described in Example 14. The detected protein migrated in the same manner as observed with human I domain protein.

B. Preparation of monoclonal antibodies against rat Od-I domain/HuIgG4 fusion proteins

Mice were individually immunized with 50 µg of purified rat dd-I domain/HuIgG4 fusion protein previously emulsified in an equal volume of Freund's complete adjuvant (FCA) (Sigma). Approximately 200 µl of the antigen/adjuvant preparation was injected at four sites in the back and sides of each of the mice.

2 weeks later, the mice received a booster injection of

100 µl rat dd-1 domain/HuIgG4 antigen (50 µg/mouse) which was previously emulsified in an equal volume of Freund's incomplete adjuvant (FIA). After a further 2 weeks, the mice received a booster injection of 50 µg antigen in 200 µl PBS intravenously.

To evaluate serum titers in the immunized mice, retroorbital blood draws were performed from the animals 10 days after the third immunization. The blood was allowed to clot, and serum was isolated by centrifugation. The serum was used in an immunoprecipitation on biotinylated (BIP) rat splenocytes. Serum from each mouse immunoprecipitated protein bands of expected molecular weight for rat Od and rat CD18. One mouse was selected for fusion and received a booster injection a fourth time, as described above, for the third booster injection.

The hybridoma supernatants were screened by antibody capture described as follows. Immulon 4 plates (Dynatech, Cambridge, Massachusetts) were coated at 4°C with 50 µl/well goat antimouse IgA, -IgG, or -IgM (Organon Teknika) diluted 1:5000 in 50 mM carbonate buffer, pH 9.6 . Plates were washed three times with PBS containing 0.05% Tween 20 (PBST), and 50 µl of culture supernatant was added. After incubation at 37°C for 30 minutes and washing, as described above, 50 µl of horseradish peroxidase-conjugated goat anti-mouse IgG9(fc) (Jackson ImmunoResearch, West Grove, Pennsylvania) diluted 1:3500 in PBST was added. The plates were incubated as described above and washed four times with PBST. Immediately thereafter, 100 µl substrate containing 1 mg/ml o-phenylenediamine (Sigma) and 0.1 µl/ml 30% H 2 O 2 in 100 mM citrate, pH 4.5 was added. The color reaction was stopped after 5 minutes by the addition of 50 µl of 15% H 2 SO 4 . Absorbance at 490 nm was read on a Dynatech plate reader.

Supernatant from antibody-containing wells was also analyzed by ELISA with immobilized rat cta-I domain/HuIgG4 fusion protein. An ELISA with HuIgG4 antibody-coated plates served as a control for reactivity against the IgG fusion partner. Positive wells were selected for further screening by BIP on rat splenocyte lysates using techniques described below.

C. Preparation of polyclonal sera against rat aa-I domain/HuIgG4 fusion protein

Blood samples were taken from two rabbits before immunization with 100 µg of purified rat cta-1 domain/HuIgG4 fusion protein in complete Freund's adjuvant. Injections were repeated with the same dose in incomplete Freund's adjuvant (IFA) every 3 weeks. After three injections, test blood samples were drawn from the rabbits, and the collected sera were used in a standard immunoprecipitation on rat splenocyte lysates. Sera from both rabbits were determined to be immunoreactive with rat Oa. The rabbits were again given booster injections with 100 pg antigen in IFA, and the collected sera were analyzed for increased immunoreactivity with rat-cid by immunoprecipitation. The animals were given a final booster injection, and 10 days later all blood was bled and sera collected.

Rat- Qd- hiBtology

Rabbit polyclonal sera raised against the rat ad "I" domain were used in immunohistochemical staining of rat tissue sections using the technique described in Example 16. The staining pattern detected on frozen and paraffin-embedded rat spleen sections was substantially identical to the pattern observed with the antibodies against human ad with staining of individual cells throughout the red mass. The staining pattern was different from that observed with monoclonal antibodies against rat CD11a, -CD11b and -CD18. A positive staining pattern was also observed in the thymus on individual cells throughout the cortex. None of these tissues gave any signal when stained with rabbit preimmune sera.

D. Analysis of antibody specificity

Rats were euthanized by asphyxiation with CO 2 , and spleens were removed using standard surgical techniques. Splenocytes were harvested by gently pressing the spleen through a wire mesh with a 3 cm<3> syringe plunger in 20 ml RPMI. Cells were collected in a 50 ml conical tube and washed in the appropriate buffer.

Cells were washed three times in cold D-PBS and resuspended at a density of 10B to 10<9> cells in 40 ml PBS. 4 mg of NHS-biotin (Pierce) was added to the cell suspension and the reaction was allowed to proceed for exactly 15 min at room temperature. The cells were pelleted and washed three times in cold D-PBS. The cells were resuspended at a density of 108 cells/ml in cold lysis buffer (1% NP40; 50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 2 mM CaCl; 2 mM MgCl; 1:100 solution of pepstatin, leupeptin and aprotinin, added just before addition to the cells; and 0.0001 g of PMSF crystals, added just before addition to the cells). Lysates were vortexed for approx. 30 seconds, incubated for 5 minutes at room temperature and incubated for an additional 15 minutes on ice. Lysates were centrifuged for 10 min at 10,000 x g to pellet the insoluble material. Supernatant was collected in a new tube and stored at between 4 °C and -20 °C. 1 ml of cell lysate was pre-cleared by incubation with

200 µl of a protein A-Sepharose slurry {Zymed) overnight at 4 °C. Aliquots of 50 µl/tube of pre-cleared lysate were transferred to Eppendorf tubes for each antibody to be tested. 25 µl of polyclonal serum or 100-500 µl of monoclonal antibody supernatant was added to the pre-B-clarified lysates, and the resulting mixture was incubated for 2 hours at 4°C with agitation. 100 µl of rabbit anti-mouse IgG (Jackson) bound to protein A-Sepharose beads in a PBS slurry was then added, and incubation was continued for 30 min at room temperature with agitation. The beads were pelleted by gentle centrifugation and washed three times with cold wash buffer (10 mM HEPES, 0.2 M NaCl, and 1% Triton-X-100). Supernatant was removed by aspiration, and 20 µl of 2 x SDS sample buffer containing 10% β-mercaptoethanol was added. The sample was boiled for 2 minutes in a water bath, and the sample was applied to a 5% SDS-PAGE gel. After separation, the proteins were transferred to nitrocellulose overnight at constant current. The nitrocellulose filters were blocked with 3% BSA in TBS-T for 1 h at room temperature, and the blocking buffer was removed. A 1:6000 dilution of streptavidin-HRP conjugate (Jackson) in 0.1% BSA-TBS-T was added, and incubation was continued for 30 min at room temperature. The filters were washed three times for 15 min with TBS-T and autoradiographed using the Amersham ECL kit according to the manufacturer's suggested protocol.

E. Preparation of monoclonal antibodies against unabridged rat dd protein

Purification of rat Qd protein

Rat-Od was purified from rat splenocytes to prepare an immunogen to generate anti-rat-Od monoclonal antibodies. Spleen from approx. 50 normal female Lewis rats, 12-20 weeks old, were collected and a single cell suspension was formed from the tissue by forcing it through a fine wire mesh. Red blood cells were removed by lysis in buffer containing 150 mM NH 4 Cl; 10 mM KHCO 3 ; 0.1 mM EDTA, pH 7.4; and remaining leukocytes were washed twice with phosphate-buffered saline (PBS). The splenocytes were pelleted by centrifugation and lysed in buffer containing 50 mM Tris, 150 mM NACl, 2 mM CaCl2/2 mM MgCl2, 10 mM PMSF, leupeptin, pepstatin and 1% Triton X-100. Splenocyte lysis was performed on ice for 30 minutes with 1 ml of lysis buffer per 5 x IO<8> splenocytes. Insoluble material was removed by centrifugation.

CDlla, CDllb and CDllc were removed from the spleen lysate by immunoprecipitation as follows. 750 µl of a protein A-Sepharose slurry was incubated with 2 mg of rabbit anti-mouse immunoglobulin at 4°C for 30 minutes. The rabbit anti-mouse protein A-Sepharose was washed three times with lysis buffer and suspended in a final volume of 1.5 ml of lysis buffer. Approximately 200 µg of each of rat fetal integrin-specific monoclonal antibodies 515 F (specific for rat CDlla), OX-42 (specific for rat CDllb) and 100g (specific for rat CDllc) were added to 50 ml of the rat spleen lysate. After 30 minutes of incubation at 4°C, 500 µl of rabbit anti-mouse protein A-Sepharose was added to the spleen lysates and mixed by "end-over-end" rotation for 30 minutes at 4°C. The lysate was centrifuged at 2500 x g for 10 minutes to pellet CDlla, CDllb and CDllc bound to rabbit anti-mouse protein A-Sepharosen, and the supernatant was transferred to a clean 50ml centrifuge tube. Immunoprecipitation with antibodies 515F, OX-42 and 100g was repeated twice more to ensure complete removal of CDlla, CDllb and CDllc.

β2 integrins remaining in the lysate were isolated using affinity purification. Approximately 250 µl of a slurry of anti-rat CDia monoclonal antibodies 20C5B, conjugated to CNBr-Sepharose, was added to the lysates and mixed by "end-over-end" rotation for 30 minutes at 4°C. Antibody/antigen complexes were pelleted by centrifugation at 2500 x g for 10 min, and the pellet was washed three times with lysis buffer before being stored at 4°C.

Immunization of Armenian hamsters

Armenian hamsters aged 6-8 weeks were initially immunized with approx. 50 µg of a recombinant protein consisting of the I domain of rat aa fused to the human IgG4 heavy chain emulsified in complete Freund's adjuvant. Primary immunization was followed by subsequent immunizations with rat Ad-I domain/HuIgG4 emulsified in incomplete Freund's adjuvant on days 14, 33 and 95. Two separate fusions, designated 197 and 199, were then performed. 4 days before fusion 197 (day 306) one hamster was administered a combination of rat aa protein purified from splenocytes and CHO cells transfected with rat Od The fusion boost injection was given 3 days before fusion (day 307) with purified rat -Od protein and aa transfected pea CHO cells. Rotecia-transfected CHO cells were prepared as described below.

A gene segment encoding truncated rat aa protein was inserted into the pDC1 vector and transfected by electroporation into CHO cells together with a human CD18 pRC construct. Transfected cells were grown in the presence of hypoxanthine to select cells successfully transfected with the pRC construct, and in the presence of g418 to select cells transfected with the pDC1 construct. After 3 weeks, the cells were stained with the rat-da-spes in five rabbit polyclonal sera and sorted by FACS. A small percentage of cells expressing the highest levels of surface aa (about 3% of the total population) were collected and further expanded. FACS selection was repeated several times to provide a cell population with high levels of aa surface expression.

The aa-transfected cells were also characterized by flow cytometry using a rat aa-specific polyclonal serum and a human CD18-specific monoclonal antibody, TS1.18.1. The results confirmed that the transfected CHO cells expressed high levels of both rat Ad and human CD18.

Finally, Ad and CD18 expression in the cells was evaluated by immunoprecipitation. A rat aa-specific rabbit polyclonal serum was found to immunoprecipitate proteins of two distinct molecular weights: the higher molecular weight protein(s) was approx. 170 kD, and the lower molecular weight protein(s) was 95 kD. These findings were consistent with the expression of a heterodimeric complex of rat Ad/human CD18 on the surface of the

transfected CHO cells.

On the day of fusion, the spleen was removed, and a single-cell suspension was formed by rubbing the tissue between the matte-ground ends of two microscope slides immersed in serum-free RPMI 1640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate,

100 units/ml penicillin and 100 ug/ml streptomycin (RPMI)

{Gibco, Canada). The cell suspension was filtered through a sterile 70-mesh Nitex cell filter (Becton Dickinson, Parsippany, New Jersey) and washed twice by centrifugation at 200 x g for 5 min and resuspending the pellet in 20 ml of serum-free RPMI. Thymocytes taken from three untreated Balb/c mice were prepared in a similar manner. NS-1 myeloma cells, maintained in log phase in RPMI with 10% Fetalclone serum (FBS)

(Hyclone Laboratories, Inc., Logan, Utah) for 3 days before fusion, was centrifuged at 200 x g for 5 min, and the pellet was washed twice, as described previously.

Approximately 1.15 x 10<8> spleen cells were combined with 5.8 x 10<7> NS-1 cells and centrifuged, and the supernatant was removed by aspiration. The cell pellet was resuspended by tapping the tube, and 7 ml of 37 °C PEG 1500 (50% in 75 mM HEPES, pH 8.0)

(Boehringer Mannheim) was added with stirring during

1 minute, followed by the addition of 14 ml of serum-free RPMI over 7 minutes. An additional 8 ml of RPMI was added and the cells were centrifuged at 200 x g for 10 min. The supernatant was removed, and the pellet was resuspended in 200 ml RPMI containing 15% FBS, 100 mM sodium hypoxanthine, 0.4 mM aminopterin, 16 mM thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim) and 1 .5 x IO<6> thymocytes/ml. The suspension was transferred to ten 96-well, flat-bottomed tissue culture plates (Corning, UK) in an amount of 200 ug/well, and the cells were supplied with nutrients on days 4, 5, 6 and 7 after fusion by aspiration of approx. 100 µl from each well with an 18 G needle (Becton Dickinson) and addition of 100 µl of diffusion medium, described above, except that thymocytes were missing.

On day 10, supernatants from the fusion wells were screened by flow cytometry for reactivity to rat aa/human CD18-transfected CHO cells. About 5 x 10<5 >rat Od-transfected CHO cells were suspended in 50 µl RPMI containing 0.2% FBS and 0.05 sodium azide, and added to about 100 µl hybridoma culture supernatant in 96-well, round-bottom plates. Positive controls for staining included rabbit anti-ad polyclonal sera and TS1/18 (antihuman CD18). Cells were incubated for 30 min on ice, washed three times in FACS buffer (RPMI, 2.0% FBS, 0.05% Na-azide) and incubated for 30 min on ice with a FITC-conjugated goat anti-hamster antibody (Jackson ImmunoResearch Labs) at a final dilution of 1:200 in FACS buffer. Cells were washed three times in FACS buffer and resuspended in 200 ml FACS buffer. Samples were analyzed with a Becton Dickinson FACScan analyzer. To ensure that wells with positive clones were specific for rat aa, the screening was repeated with untransfected CHO cells. Wells that met the criteria for reaction with rat aa-CHO transfectants and not the untransfected CHO cells were cloned.

After primary screening, cells from positive wells were first cloned by 2-fold dilution and then by limiting dilution in RPMI, 15% FBS, 100 mM sodium hypoxanthine, 16 mM thymidine and 10 units/ml IL-6. In the limiting dilution step, the percentage of wells showing growth was determined and clonality was pre-determined using a Poisson distribution analysis. Wells showing growth were analyzed by FACS after 10-12 days. After the last cloning, positive wells were expanded in RPMI and 11% FBS. Cloning yielded one culture judged positive by these criteria, and from this clone four separate sub-clones, designated 197A-1, 197A-2, 197A-3 and 197A-4, were expanded.

Prior to fusion 199, a second hamster was given a booster injection on day 307 with 2.3 x 10<6> rat Oa-transfected CHO cells. Two final immunizations were administered 4 days before fusion (day 334) and again 3 days before fusion (day 335). The boost injection on day 334 consisted of 2 x 10<6 >rat aa-transfected CHO cells and 200 µl of purified rat aa bound to Sepharose (described previously), administered by intraperitoneal injection. The boost injection on day 335 consisted of 5 x 10<6> rat aa-transfected CHO cells, also administered by intraperitoneal injection. The fusion and screening protocols for fusion 199 were identical to fusion 197, and three hybridomas, designated 199A, 199H, and 199M, with supernatant reactive with rat aa, were identified and cloned. The hybridoma designated 199M was deposited on March 1, 1996 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, and given accession number HB12058.

Characterization of monoclonal antibodies against rat Od

To characterize the anti-rat Od antibodies, biotin-labeled spleen lysates were prepared, as described in Example 18, Section D, above. Lysates were pre-cleared before use in immunoprecipitations. 50 µg/ml normal mouse immunoglobulin was first added to the lysate and the resulting solution was mixed by "end-over-end" rotation for 30 minutes at 4°C. 75 µl of a slurry of protein A-Sepharose coated with rabbit anti-mouse immunoglobulin was added and the mixture continued by "end-over-end" rotation for 30 minutes. The rabbit antimouse coated protein A beads were pelleted by centrifugation at 15,000 rpm in a benchtop microcentrifuge for 5 minutes at 4°C, and the supernatant was collected. The pelleted material was discarded.

For each cloned hybridoma, approx. 300 ul of supernatant placed in Eppendorf microcentrifuge tubes, and to this was added 30 ul of 10% Triton X-100, 30 ul of a 100x stock solution of pepstatin, leupeptin and aprotinin, 100 pg of PMSF crystals and 50 ul of pre-cleared biotinylated rat spleen lysate. The samples were gently vortexed and placed on an end-over-end rotator at 4°C for 30 minutes. A control sample was prepared by adding 10 mg/ml of a rabbit anti-rat Od-specific polyclonal antibody to 50 µl of rat spleen lysate.

After incubation for 30 minutes, 75 µl of protein A-Sepharose beads in a PBS slurry were added to each sample and incubated with "end-over-end" rotation at 4°C for 30 minutes. The protein A-coupled beads were pelleted by centrifugation at 15,000 rpm in a benchtop microcentrifuge for 5 minutes at 4°C, and the supernatant was collected. The pelleted beads were washed sequentially with a series of 1 ml detergent washes which

follows: buffer No. 1 containing 10 mM Tris, 400 mM NaCl,

1.0% Triton X-100, pH 8.0; buffer #2 containing 10 mM Tris, 400 mM NaCl, 0.5% Triton X-100, pH 8.0; buffer #3 containing 10 mM Tris, 400 mM NaCl, 1.0% Triton X-100,

0.1% deoxycholate, pH 8.0; and buffer #4 containing 10 mM Tris, 400 mM NaCl, 0.5 M LiCl 2 , pH 8.0. A final wash was performed with wash buffer No. 1. The beads were gently vortexed between each wash and pelleted using a tabletop microcentrifuge. Supernatants were removed using a transfer pipette, and after the final wash, any remaining buffer was removed from the beads with a Hamilton syringe. A 50 µl aliquot of SDS sample buffer containing the dyes bromophenol blue and pyronin Y and β-mercaptoethanol, at a final concentration of 10%, was added to each pellet. The mixture was vortexed vigorously for 1-2 minutes and incubated at room temperature for 5-10 minutes. Samples were centrifuged for 5 min at 15,000 rpm in a tabletop microcentrifuge at 4°C, and released protein was collected and transferred to a new microcentrifuge tube. Aliquots from each sample were boiled for 4 min in a water bath before running on 7.5% SDS-PAGE gels. After separation by PAGE, proteins were transferred to nitrocellulose filters for 1 hour at 200 mA, and the filters were blocked in a solution of 3% BSA/PBS-T overnight at 4 °C. A solution of 0.1% BSA-TBS-T containing a 1:6000 dilution of streptavidin-OPD was added to each filter, and incubation was allowed to proceed for 1 hour at room temperature. Filters were washed five times for 10 min in TBS-T and developed using Amersham's ECL kit according to the manufacturer's suggested protocol.

Clone 199M was found to immunoprecipitate a heterodimeric protein. The larger protein subunit had an approximate molecular weight of 170-175 kD, which was consistent with the size of the protein immunoprecipitated by the rabbit anti-rat Od polyclonal control. A second protein with an approximate molecular weight of 95 kD was also precipitated, which is in agreement with the weight of CD18.

Example 19

Isolation of mouse cDNA clones

Isolation of a mouse dd homolog was attempted.

Interspecies cross-hybridization was performed using two PCR-provided probes: a 1.5 kb fragment corresponding to bases 522-2047 of human clone 19A2 (SEQ ID NO: 1), and a 1.0 kb rat fragment corresponding to corresponds to bases 1900-2900 in human clone 19A2 (SEQ ID NO: 1). The human probe was generated by PCR using primer pairs designated ATM-2 and 9-10.1, respectively shown in SEQ ID NO. NO. 38 and 39; the rat probe was generated using primer pairs 434L and 434R, respectively shown in SEQ ID NO. NO. 34 and 35. Samples were incubated at 94°C for 4 minutes and subjected to 30 cycles of the temperature step sequence: 94°C; 50 °C, 2 minutes; 72 °C, 4 minutes.

The PCR products were purified using the Qiagen Quick Spin kit in accordance with the manufacturer's suggested protocol, and approx. 180 ng of DNA was labeled with 200 pCi [<32>P]-dCTP using a Boehringer Mannheim Random Primer labeling kit in accordance with the manufacturer's suggested protocol. Unincorporated isotope was removed using a Centrisep Spin column (Princeton Separations, Adelphia, NJ) in accordance with the manufacturer's suggested protocol. The probes were denatured with 0.2 N NaOH and neutralized with 0.4 M Tris-HCl, pH 8.0, before use.

A mouse thymus oligo dT-primed cDNA library in AZAPII (Stratagene) was propagated at ca. 30,000 plaques per 15 cm plate. Plaque transfers on nitrocellulose filters (Schleicher & Schuell, Keene, NH) were incubated at 50°C with agitation in

1 hour in a prehybridization solution (8 ml/transfer) containing 30% formamide. Labeled human and rat probes were added to the prehybridization solution, and incubation was continued overnight at 50°C. Filters were washed twice in 2 x SSC/0.1% SDS at room temperature, once in 2 x SSC/0.1%

SDS at 37 °C and once in 2 x SSC/0.1% SDS at 42 °C. Filters were exposed with Kodak X-Omat AR film at -80°C for 27 hours with an intensifying screen.

Four plaques that gave positive signals on duplicate transfers were replated on LB medium with magnesium (LBM)/carbenicillin plates (100 mg/ml) and incubated overnight at 37°C. The plaques were blotted with Hybond filters (Amersham), probed as in the initial screening and exposed to Kodak X-Omat AR film for 24 hours at -80°C with an intensifying screen. 12 plaques giving positive signals were transferred to a low Mg<++> phage diluent containing 10 mM Tris-HCl and 1 mM MgCl 2 . Insertion size was determined by PCR amplification using T3 and T7 primers (SEQ ID NO: 13 and 14, respectively) and the following reaction conditions. Samples were incubated at 94°C for 4 minutes and subjected to 30 cycles of the temperature step sequence: 94°C, 15 seconds; 50 °C, 30 seconds and 72 °C, 1 minute. 6 samples produced distinct bands ranging in size from 300 bases to 1 kb. Phagemids were released via co-infection with the helper phage and recirculated to form Bluescript SK" (Stratagene). The resulting colonies were grown in LBM/carbenicillin (100 mg/ml) overnight. DNA was isolated with a Promega Wizard miniprep kit (Madison, WI) in accordance with the manufacturer's suggested protocol. Ecol restriction analysis of the purified DNA confirmed the molecular weights, which were detected using PCR. Inserted DNA was sequenced with Ml3 and Ml3 reverse.1 primers, respectively shown in SEQ ID NO: ID NOS 40 and 41.

Sequencing was performed as described in Example 4.

Of the 6 clones, only 2, designated 10.3-1 and 10.5-2, provided sequence information and were identical 600 bp fragments. The 600 bp sequence was 68% identical to a corresponding region of human da, 40% identical to human CDlla, 58% identical to human CDllc and 54% identical to mouse CDllb. This 600 bp fragment was then used to isolate a more complete cDNA encoding a putative mouse Od homologue.

A mouse spleen cDNA library (oligo dT" and randomly primed) in AZAPII (Stratagene) was spread at 2.5 x 10<*> phage/ 15 cm LBM plate. Plaques were blotted onto Hybond nylon transfer membranes (Amersham) , denatured with 0.5 M NaOH/1.5 M NaCl, neutralized with 0.5 M Tris base/1.5 M NaCl/11.6 M HCl and washed in 2 x SSC.DNA was cross-linked to filters using of ultraviolet irradiation.

Approximately 500,000 plaques were screened using probes 10.3-1 and 10.5-2 previously labeled as described above. Probes were added to a prehybridization solution and incubated overnight at 50°C. The filters were washed twice in 2 x SSC/0.1% SDS at room temperature, once in 2 x SSC/0.1% SDS at 37 °C and once in 2 x SSC/0.1% SDS at

42 °C. Filters were exposed with Kodak X-Omat AR film i

24 hours at -80 °C with an intensification screen. 14 plaques that gave positive signals on duplicate transfers were subjected to a second screening identical to the initial screening, except for additional final high-stringency washes in 2 x SSC/0.1% SDS at 50°C, for 0.5 x SSC/0.1% SDS at 50 °C and at 55 °C in 0.2 x SSC/0.1% SDS. The filters were exposed with Kodak X-Omat AR film at -80°C for 13 hours with an intensifying screen. 18 positive plaques were transferred to phage diluent with low Mg<++> content, and the insert size was determined by PCR amplification, as described above. 7 of the samples gave single bands ranging in size from 600 bp to 4 kb. EcoRI restriction analysis of purified DNA confirmed the sizes observed from PCR, and the DNA was sequenced with primers M13 and M13 reverse.1 (SEQ ID NO: 40 and 41, respectively).

One clone designated B3800 contained a 4 kb insertion corresponding to a region 200 bases downstream of the 5' end of the human Od-19A2 clone and including 553 bases of a 3' untranslated region. Clone B3800 showed 77% identity with et

corresponding region of human aa, 44% identity with a corresponding region of human CDlla, 59% identity with a corresponding region of human CDllc and 51% identity with a corresponding region of mouse CDllb. The second clone, A1160, was a 1.2 kb insertion that matched the 5<1> end of the coding region of human ctd ca. 12 nucleic acids downstream of the initial methionine. Clone Al160 showed 75% identity with a corresponding region of human ctd, 46% identity with a corresponding region of human CDlla, 62% identity with a corresponding region of human CDllc and 66% identity with a corresponding region of mouse CDllb.

Clone A1160, the fragment closer to the 5<1> end of human clone 19A2, is 1160 bases long and shares an overlap region with clone B3800 starting at base 205 and continuing to base 1134. Clone A1160 has an insertion of 110 bases (bases 704-814 in clone A1160) which is not present in the overlapping region in clone B3800. This insertion occurs at a putative exon-intron boundary [Fleming et al., J. Immunol., 250:480-490 (1993)] and was removed prior to subsequent ligation of clones A1160 and B3800.

Rapid amplification of the 5' cDNA end of the putative mouse Od clone

RACE-PCR [Frohman, "RACE: Rapid Amplification of cDNA Ends", in PCR Protocols: A Guide to Methods and Applications, Innis et al. (ed.), pp. 28-38, Academic Press, New York

(1990)] was used to obtain missing 5' sequences of the putative mouse Ad clone, including 5' untranslated sequence and leading methionine. A mouse spleen RACE-Ready kit (Clontech, Palo Alto, CA) was used according to the manufacturer's suggested protocol. Two antisense, gene-specific primers, A1160-RACE1 primer and A1160-RACE2 nested (SEQ ID NOS: 42 and 43), were designed to perform primary and nested PCR.

The primers, SEQ.ID. NO. 42 and 43, respectively, correspond to the regions starting 302 and 247 base pairs from the 5' end. PCR was performed as described above using the 5' anchor primer (SEQ ID NO: 44) and mouse spleen cDNA supplemented with the kit.

Electrophoresis of the PCR product revealed a band of size approx. 280 bases, which were subcloned using a TA cloning kit (Invitrogen) in accordance with the manufacturer's suggested protocol. 10 resulting colonies were cultured, and DNA was isolated and sequenced. An additional 60 bases of the 5' sequence were identified using this method, corresponding to bases 1-60 of SEQ ID NO. NO. 45.

Characteristics of the mouse cDNA and the precalculated amino acid sequence

A composite sequence of mouse cDNA encoding a putative homologue of human aa is shown in SEQ ID NO. NO. 45. Although the homology between the external domains of the human clones and the mouse clones is high, the homology between the cytoplasmic domains is only 30%. The observed variation may indicate C-terminal functional differences between the human proteins and the mouse proteins. Alternatively, the variation in the cytoplasmic domains may result from splicing variations, or it may indicate the existence of an additional pVintegrin gene(s).

At the amino acid level, the mouse cDNA predicts a protein (SEQ ID NO: 46) with 28% identity to mouse CDlla, 53% identity to mouse CDllb, 28% identity to human CDlla, 55% identity to human CDllb, 59% identity with human CDllc and 70% identity with human ad. Comparison between the amino acid sequences in the cytoplasmic domains of human ad and the putative mouse homologue indicates regions of the same length, but with differing primary structure. Similar sequence length in these regions suggests species variation rather than splice variant forms. When compared with the predicted rat polypeptide, Example 16 above, the mouse and rat cytoplasmic domains show more than 60% identity.

Example 20 Isolation of additional mouse Od cDNA clones for sequence verification

To verify the nucleic acid and amino acid sequences described in Example 19 for mouse Od, additional mouse sequences were isolated for confirmatory purposes.

Isolation of mouse cDNA by hybridization with two homologous ad probes (3' and 5<1>) was performed using both an arbitrarily primed mouse spleen library and an oligo dT-primed cDNA library in AZAPII (Stratagene). The library was spread out at 5 x 10<5> subjects per 15 cm LBM plate. Plaques were blotted onto Hybond nylon membranes (Amersham), and the membranes were denatured (0.5 M NaOH/1.5 M NaCl), neutralized (0.5 M Tris base/1.5 M NaCl/11.6 M HCl , and washed (2 x SSC-saline).DNA was cross-linked to filters using ultraviolet irradiation.

Probes were generated using primers described below in a PCR reaction under the following conditions. Samples were held at 94°C for 4 minutes and then subjected to 30 cycles of the temperature step sequence (94°C for 15 seconds; 50°C for 30 seconds; 72°C for 1 minute, in a Perkin-Eimer 9600 thermocycler).

The 3' probe was approx. 900 bases long and spanned a region from nucleotides 2752 to 3651 (in SEQ ID NO: 1) (5'->3') and was produced with primers 11.b-1/2FOR11 and 11.b-1/2REV2, as shown in SEQ.ID respectively. NO. 69 and 74. This probe was used in a first set of suctions.

The 5<1->probe was approx. 800 bases long and spanned a region from nucleotides 149 to 946 (in SEQ ID NO: 1) (5'->3') and was produced with primers 11.b-1/2F0R1 and 11.a-1/1REV1, as shown in SEQ.ID respectively. NO. 50 and 85. This probe was used in a second set of suctions.

In a third set of absorptions, both probes described above were used together on the same plates.

Approximately 500,000 plaques were screened using the two probes described above, which were labeled in the same manner as described in Example 17. Labeled probes were added to a prehybridization solution containing formamide and incubated overnight at 50°C. Filters were washed twice in 2 x SSC/0.1% SDS at room temperature (22°C). A final wash was performed in 2 x SSC/0.1% SDS at 50 °C. Autoradiography was performed for 19 hours at -80°C on Kodak X-Omat AR film with an intensifying screen. 13 plaques that gave positive signals on at least two aspirates were subjected to a second screening performed as described for the initial screening, except that both the 3'- and 5'-labeled probes were used for hybridization and an additional final wash was incorporated using 2 x SSC/0.1% SDS at 65 °C. Autoradiography was performed as described above for 2.5 hours. 13 plaques (designated MS2P1-MS2P13) that gave positive signals were transferred to a low Mg<++> phage diluent. Insert size was determined by PCR amplification (Perkin-Eimer 9600 thermocycler) using T3 and T7 primers annealing to Bluescript phagemid in ZAPII (sequence described previously) under the same conditions as shown above. Band sizes ranged from 500 bases to 4 kb. Phagemids were isolated, prepared and sequenced with M13 and M13 reverse.1 primers (SEQ ID NO: 40 and 41, respectively). 5 of the 13 clones, MS2P-3, MS2P-6, MS2P-9, MS2P-12 and MS2P-13, were sequenced and together represented an area from approx. base 200 at the 5' end to approx. 300 bases past a first stop codon at the 3' end.

Automated sequencing was performed as described in Example 4 by first using M13 and M13-reverse.1 primers (SEQ ID NO: 40 and 41, respectively) to sequence the ends of each clone and determine the position relative to construct no. 17 (SEQ ID NO. 45). Each clone was then completely sequenced using the appropriate primers (listed below) for that particular region.

Sequences were edited, aligned and compared to a previously isolated mouse Ad sequence {construct no. 17, SEQ ID NO. NO. 45).

Alignment of the new sequences revealed an 18 base deletion in construct #17 starting at nucleotide 2308; the deletion did not cause a shift in the reading frame. Clone MS2P-9, sequenced as described above, also revealed the same 18 base deletion. The deletion has been observed to occur in 50% of mouse clones that include the region, but has not been detected in rat or human dd clones. The 18-base deletion is characterized by a 12-base palindromic sequence, AAGCAGGAGCTCCTGTGT (SEQ ID NO: 91). This inverted repeat in the nucleic acid sequence is self-complementary and can form an outpouch and cause cleavage during reverse transcription. The mouse aa sequence including the additional 18 bases is shown in SEQ ID NO. NO. 52; the deduced amino acid sequence is shown in SEQ ID NO. NO. 53.

Example 21

In si tu - hybridizations in mice

Tissue distribution was then determined for mouse ctd to provide a comparison with the distribution in humans, described in Example 6.

A single-stranded 200 bp mRNA probe was generated from a DNA template, corresponding to nucleotides 3460-3707 in the cytoplasmic tail region of mouse cDNA, by in vitro RNA transcription incorporating <3>SS-UTP (Amersham) .

Whole mouse embryos (removed on days 11-18 post-fertilization) and various mouse tissues, including spleen, kidney, liver, intestine, and thymus, were hybridized in situ with the radiolabeled, single-stranded mRNA probe.

Tissues were sectioned at 6 µm thicknesses, mounted on Vectabond (Vector Laboratories, Inc., Burlingame, CA) coated slides and stored at -70°C. Before use, the slides were placed at 50 °C for approx. 5 minutes. Sections were fixed in 4% paraformaldehyde for 20 min at 4 °C, dehydrated with an increasing ethanol gradient (70-95-100%) for 1 min at 4 °C for each concentration, and air-dried for 30 min at room temperature. Sections were denatured for 2 min at 70°C in 70% formamide/2x SSC, rinsed twice in 2x SSC, dehydrated with the ethanol gradient described above, and air-dried for 30 min. Hybridization was performed overnight (12-16 h) at 55 °C in a solution containing <35>S-labeled riboprobes at 6 x 10<5> cpm/section and diethyl pyrocarbonate (DEPC)-treated water to give a final concentration of 50% formamide, 0.3 M NaCl, 20 mM Tris-HCl, pH 7.5, 10% dextran sulfate, 1x Denhardt's solution, 100 mM dithiothreitol (DTT), and 5 mM EDTA. After hybridization, the sections were washed for 1 h at room temperature in 4 x SSC/10 mM DTT, for 40 min at 60 °C in 50% formamide/2 x SSC/10 mM DTT, for 30 min at room temperature in 2 x SSC and for 30 minutes at room temperature in 0.1 x SSC. Sections were dehydrated, air-dried for 2 hours, coated with Kodak NTB2 photographic emulsion, air-dried for 2 hours, developed (after storage at 4°C in complete darkness) and counterstained with hematoxylin/eosin.

Spleen tissue showed a strong signal mainly in the red mass. This pattern is consistent with the pattern of tissue macrophage distribution in the spleen, but does not exclude other cell types.

Example 22

Generation of mouse expression constructs

To construct an expression plasmid including mouse cDNA sequences exhibiting homology to human eid, inserts from clones A1160 and B3800 were ligated. However, prior to this ligation, a 5' leader sequence that included an initial methionine was added to clone A1160. A primer designated "5'-PCR leader" (SEQ ID NO: 47) was designed to contain: (1) identical non-specific bases in positions 1-6, which enable cleavage; (2) a BamHI site (underlined in SEQ ID NO: 47) from positions 7 to 12 to facilitate subcloning into an expression vector; (3) a consensus Kozak sequence from positions 13 to 18; (4) a signal sequence that includes a codon for an initial methionine (in bold in SEQ ID NO: 47); and (5) an additional 31 bases of specifically overlapping 5' sequence from clone Al160 to allow primer annealing. A second primer designated "3' end fragment" (SEQ ID NO: 48) was used with primer "5•-PCR leader" to amplify the insert from clone A1160.

The resulting PCR product was not digested with BamHI, suggesting that an insufficient number of bases were located upstream of the restriction site, preventing recognition by the enzyme. The length of the "tail" sequence preceding the BamHI site in the 5' primer (SEQ ID NO: 47) was increased, and the PCR was repeated on the amplification product from the first PCR. A 5' primer, designated mAD.5'.2 (SEQ ID NO: 49), was designed with additional non-specific bases in positions 1-4 and an additional 20 bases that specifically overlapped the previously used "5<1-> PCR Leader" primer sequences.

Primers "mAD.5'.2" and "3'-end fragment" were used together in PCR with the product from the first amplification as template. A resulting secondary PCR product was subcloned into plasmid pCRtmll (Invitrogen) according to the manufacturer's suggested protocol and transformed into competent one shot cells (Invitrogen). One clone containing the PCR product was identified by restriction enzyme analysis using BamHI and EcoRI and sequenced. After the sequence was verified, the insert was isolated by digestion with BamHI and EcoRI and gel purified.

The insert from clone B3800 was isolated by digestion with EcoRI and Noti, gel purified and added to a ligation reaction that included the enlarged A1160-BamHI/EcoRI fragment. The ligation was allowed to proceed for 14 h at 14 °C. Vector pcDNA.3 (Invitrogen), digested with BamHI and Noti, was added to the ligation reaction with additional ligase, and the reaction was continued for an additional 12 h. An aliquot of the reaction mixture was transformed into competent E. coli cells, the resulting colonies were grown, and one positive clone was identified by PCR analysis with primers 11.b-1/2F0R1 and 11.b-1/2REV11 (SEQ ID NO: Nos. 50 and 51). These primers bridge the A1160 and B3800 fragments, so detection of an amplification product therefore indicates that the two fragments were ligated. The sequence of the positive clone was verified with the primers shown in SEQ ID NO.

NO. 50 and 51, which amplify from base 100 to 1405 after the initial methionine.

Example 23

Construction of a "knock-out" mouse

To make a more accurate assessment of the immunological role of the protein encoded by the putative mouse oa cDNA, a "knock-out" mouse is created in which the genomic DNA sequence encoding the putative oa homologue is interrupted of homologous recombination. The significance of the protein coded for by the interrupted gene is thereby assessed by the absence of the coded protein. Generation of knock-out mice is described by Deng et al., Mol. Cell. Biol., 13:2134-2140 (1993).

The design of such a mouse starts with the construction of a plasmid containing sequences to be "knocked out" by means of homologous recombination cases. A 750 base pair fragment of mouse cDNA (corresponding to nucleotides 1985-2733 in SEQ ID NO: 45) was used to identify a mouse genomic sequence encoding the putative mouse cid homologue from an AFIXII genome library. Primary screening resulted in 14 positive plaques, of which 7 were confirmed by secondary screening. Liquid lysates were obtained from two of the plaques that gave the strongest signal, and DNA was isolated using conventional methods. Restriction mapping and Southern analysis confirmed the authenticity of one clone, designated 14-1, and the inserted DNA was isolated by digestion with Noti. This fragment was cloned in Bluescript SKII<+>.

To identify a restriction fragment of approx. 9-14 kb, which is a length reported to optimize the probability of homologous recombination events, Southern hybridization was performed with the 750 bp cDNA probe. Prior to hybridization, a restriction map was constructed for clone 14-1. A 12 kb fragment was identified as a possible candidate, and this fragment was subcloned into pBluescript SKU<*> at a position where mouse DNA is flanked by cassettes encoding thymidine kinase. Further analysis of this clone with an I-domain probe (corresponding to nucleotides 454-1064 in SEQ ID NO: 45) indicated that the clone did not contain sequences that coded for I-

the domain.

Using the same I-domain probe, the AFIXII genome library was rescreened. First, 6 positive clones were detected, of which one remained positive in secondary screening. DNA isolated from this clone reacted strongly in Southern analysis with an I-domain probe. However, no reactivity was detected using the original 750 bp probe, indicating that this clone included regions 5' to nucleotides 1985-2773 in SEQ ID NO. NO. 45.

The lack of hybridization to the 750 bp probe could alternatively suggest that the clone was another member of the integrin family of proteins. To determine whether this explanation was plausible, the 13 kb insert was subcloned into pBluescript SKII<+>. Purified DNA was sequenced using primers corresponding to Od-I domain nucleic acid sequences 441-461, 591-612, 717-739 and reverse 898-918 in SEQ ID NO: NO. 52. Sequence information was obtained using only the first 4441-4461 primer, and only the most 5' exon of the I domain was efficiently amplified. The rest of the I domain was not amplified. The resulting clone therefore comprised exon 6 of the mouse Od gene and intron sequences to the 3' and 5' ends of the exon. Exon 7 was not represented in the clone. After sequencing, a construct containing neomycin resistance genes and thymidine kinase genes is formed.

The neomycin resistance (neor) gene is inserted into the resulting plasmid in a manner that interrupts the protein coding sequence of the mouse genomic DNA. The resulting plasmid therefore contains a neo gene in the mouse genomic DNA sequences, the entire gene being located within a thymidine kinase coding region. Plasmid construction in this manner is required to favor homologous recombination over random recombination [Chisaka et al., Nature, 355:516-529 (1992)].

Example 24

Cloning of the rabbit ag construct and screening of the rabbit cDNA library

Identification of human Oa homologues in rats and mice led to investigation of the presence of a rabbit homologue which would be useful in rabbit models of human disease states described below.

Poly A<+->RNA was prepared from a whole rabbit spleen using the Invitrogen FastTrack kit (San Diego, CA) in accordance with the manufacturer's suggested protocol and kit reagents. From 1.65 g of tissue, 73 pg of poly A<+->RNA was isolated. Rabbit spleen RNA was used to construct a ZAP cDNA expression library using a kit from Stratagene (La Jolla, CA). Resulting cDNA was directionally cloned into EcoRI and XhoI sites in the A-arms of a pBK-CMV phagemid vector, Gigapack II Gold (Stratagene) was used to package the A-arms into phage particles. The resulting biblio-tektite was estimated at approx. 8 x IO<5> particles, with an average insertion size of 1.2 kb.

The library was amplified once by spreading for confluent plaque growth, and cell lysate was collected. The amplified library was propagated with E. coli at approx. 30,000 plaque-forming units (pfu) per 150 mm plate, and the resulting mixture was incubated for 12-16 hours at 37°C to allow plaque formation. Phage DNA was transferred to Hybond N<+->nylon membranes (Amersham, Arlington Heights, Illinois). The membranes were hybridized with a mixture of two randomly primed, radiolabeled mouse aa PCR DNA probes. The first probe was generated from a PCR product spanning nucleotides 149-946 of SEQ ID NO. NO. 52. The second probe was from a PCR product that spanned nucleotides 2752-3651 in SEQ ID NO. NO. 52. The probes were labeled by random priming (Boehringer Mannheim Random Primed DNA labeling kit) and the reaction mixture was passed through a Sephadex G-50 column to remove unincorporated nucleotides. The hybridization solution was composed of 5 x SSPE, 5 x Denhardt's, 1% SDS, 40% formamide and the labeled probes at 1 x 10<6> dpm/ml. Hybridization was performed at 42°C for 16-18 hours. Filters were thoroughly washed in 2 x SSPE/0.1% SDS at room temperature and exposed to X-ray film to visualize any hybridizing plaques.

Two clones with significant sequence homology to human aa were identified. Clone no. 2 had a length of approx. 800 bp and was mapped to the 5* end of human cia. Clone #2 includes an initiating methionine and a complete leader sequence. Clone no. 7 was approx. 1.5 kb and includes an initiating methionine. The 5' end of clone #7 overlapped the end of clone #2, but the B 3<*> sequences terminated at a point outside the I domain sequences. Clone #7 was completely sequenced using the primer walking method. The deduced nucleotide and amino acid sequences for clone no. 7 are shown in SEQ ID NO: 7, respectively. NO. 100 and 101.

The predicted N-terminal amino acid sequence of rabbit cid, as determined from clones #2 and 7, indicated a protein with 73% identity to human ctd, 65% identity to mouse Od, and 58% identity to mouse CDllb, human CDllb and human CDllc. The nucleic acid sequence for clone No. 2 is shown in SEQ ID NO. NO. 92; the predetermined amino acid sequence is shown in SEQ ID NO. NO. 93.

Isolation of an untruncated rabbit aa cDNA was attempted using labeled rabbit clone #7 and rescreening the cDNA library from which the fragment originated. Twenty-five additional clones were identified, of which one, designated clone 49, was determined to be the largest. Clone 49 was completely sequenced using the nested deletion technique. The nucleotide sequences and amino acid sequences for clone 49 are shown in SEQ ID NO: 1, respectively. NO. 102 and 103. Because clones #7 and 49 did not overlap, oligonucleotides were designed to be used as primers in a first-strand rabbit spleen cDNA PCR to isolate the missing sequence.

The relationship between the assumed amino acid sequence in these two partial clones and the sequence in other leukointegrins is described in table 1.

Isolation of a rabbit α& clone allows expression of the protein, either on the surface of transfectants or as a soluble, unabridged or truncated form. This protein is then used as an immunogen for the production of monoclonal antibodies for use in rabbit models of human disease states.

Example 25

Animal models for determining the therapeutic applicability of Od

Immunohistological data from dogs and in situ hybridization of rats and mice have determined that in the spleen ctd is expressed primarily by macrophages that are present in the red pulp and in lymph glands, then found in medullary cords and sinuses. The expression pattern is remarkably similar to that reported for two mouse antigens defined by the monoclonal antibodies F4/80 and SK39. Although biochemical characterization of these mouse antigens has shown them to be distinct from dd, it is very likely that aa defines the same

macrophage subsets such as the mouse F4/80 and SK39 antigens.

In mice, SK39-positive macrophages have been identified in the red pulp of the spleen, where they may participate in the clearance of foreign materials from circulation, and in lymph gland marrow [Jutila et al., J. Leukocyte Biol., 54:30-39 (1993)]. SK39-positive macrophages have also been reported at sites of both acute and chronic inflammation. Furthermore, monocytes recruited to thio-glycollate-inflamed peritoneal cavities also express the SK39 antigen. These findings collectively suggest that if SK39<+-> cells are also o.d+, then these cells are responsible for the clearance of foreign materials in the spleen and participate in inflammation where macrophages play a significant role.

Although the function of α<i remains unclear, other more well-characterized β2 integrins have been shown to participate in many different adhesion events that facilitate cell migration, enhance phagocytosis, and promote cell-cell interactions, events that all lead to upregulation of inflammatory processes. It is therefore plausible that disruption of the normal Od function can also disrupt inflammation where macrophages play a significant role. Such an anti-inflammatory effect may result from: i) blocking macrophage recruitment to sites of inflammation, ii) preventing macrophage activation at the site of inflammation, or iii) disrupting macrophage effector functions that damage normal host tissue, either by specific autoimmune responses or as a result of bystander cell damage.

Disease states where there is evidence that macrophages play a significant role in the disease process include multiple sclerosis, arthritis, graft atherosclerosis, some forms of diabetes and inflammatory bowel disease. Animal models discussed below have been shown to reproduce many of the aspects of these human disorders. Inhibitors of Od function are tested in these model systems to determine whether the potential exists for treating the corresponding human diseases.

A. Transplant arteriosclerosis

Heart transplantation is now the accepted form of therapeutic intervention for some types of end-stage heart disease. As the use of cyclosporine A has increased the 1-year survival rate to 80%, the development of progressive graft arteriosclerosis has emerged as the main cause of death in heart transplant patients who survive the first year. Recent studies have found that the incidence of significant transplant arteriosclerosis 3 years after a heart transplant is in the range of 36-44% [Adams et al., Transplantation, 53:1115-1119 (1992); Adams et al.. Transplantation, 56:794-799 (1993)].

Transplant arteriosclerosis typically consists of diffuse, occlusive, internal lesions that affect the entire coronary artery wall and are often accompanied by lipid deposition. Although the pathogenesis of transplant arteriosclerosis remains unknown, it is probably related to the histocompatibility between donor and recipient, and is immunological in nature. Histologically, the areas of internal thickening consist primarily of macrophages, although T cells can occasionally be seen. It is therefore possible that macrophages expressing ctd may play a significant role in the induction and/or development of transplant arteriosclerosis. In such a case, monoclonal antibodies or low molecular weight inhibitors (eg soluble ICAM-R) with Od function can be given prophylactically to individuals who have received heart transplants and who are at risk of developing transplant arteriosclerosis.

Although atherosclerosis in heart transplants is the greatest threat to life, transplant arteriosclerosis can also be observed in other solid organ transplants, including kidney and liver. Therapeutic use of Od blocking agents can prevent graft arteriosclerosis in other organ transplants and reduce complications resulting from transplant failure.

One model of transplant arteriosclerosis in the rat involves heterotopic cardiac allografts transplanted through minor histocompatibility barriers. When Lewis heart allografts are transplanted into MHC class I and II compatible F-344 recipients, 80% of allografts survive at least 3 weeks, while 25% of grafts

survive indefinitely. During this minor degree of transplant rejection, arteriosclerosis lesions form in the donor heart. Arterial lesions in 120-day-old allografts typically have a diffuse, fibrotic intimal thickening that is indistinguishable in appearance from transplant arteriosclerotic lesions found in rejecting human cardiac allografts.

Rats are transplanted with hearts that are mismatched with respect to minor histocompatibility antigens, e.g. Lewis in the F-344. Monoclonal antibodies specific for rat Od or small molecule inhibitors of a<j are periodically given to transplant recipients. The treatment is expected to reduce the incidence of transplant arteriosclerosis in non-failing donor hearts. Treatment of rats with cid monoclonal antibodies or low molecular weight inhibitors need not be limited to prophylactic treatments. Blockade of ctd function is also expected to reduce macrophage-mediated inflammation and allow reversal of arterial damage in the graft.

B. Atherosclerosis in rabbits fed cholesterol

Rabbits that were fed an atherogenic diet containing a cholesterol supplement for approx. 12-16 weeks, developed internal lesions covering most of the inner surface of the ascending aorta [Rosenfeld et al., Arteriosclerosis, 7:9-23 (1987); Rosenfeld et al., Arteriosclerosis, 7:24-34

(1987)]. The atherosclerotic lesions observed in these rabbits are similar to the lesions observed in humans. Lesions contain large numbers of T cells, most of which express CD45RO, a marker associated with memory T cells. About half of the infiltrating T cells also express MHC class II antigen, and some express the IL-2 receptor, suggesting that many of the cells are in an activated state.

A feature of the atherosclerotic lesions found in

cholesterol-fed rabbits, but apparently absent in rodent models, is the accumulation of foam cell-rich lesions. Foam cell macrophages are thought to result from the uptake of oxidized

low-density lipoprotein (LDL) by specific receptors. Oxidized LDL particles have been found to be ■toxic to some cell types, including endothelial cells and smooth muscle cells. The uptake of potentially toxic, oxidized LDL particles by macrophages serves as an irritant and drives macrophage activation and contributes to the inflammation associated with atherosclerotic lesions.

Once monoclonal antibodies have been formed against the rabbit, cholesterol-fed rabbits are treated. Treatments include prophylactic administration of dd monoclonal antibodies or low molecular weight inhibitors to demonstrate that aa<+->macrophages are involved in the disease process. Further investigations will show that monoclonal antibodies against ad or low molecular weight inhibitors are able to reverse blood vessel damage detected in rabbits fed an atherogenic diet.

C. Insulin-dependent diabetes

BB rats spontaneously develop insulin-dependent diabetes at 70-150 days of age. Using immunohistochemistry, MHC class II<+>,EDl<+->macrophages can be demonstrated to infiltrate the islet cells early in the disease. Many of the macrophages appear to be engaged in phagocytosis of degraded cell material or normal cells. As the disease progresses, there are large numbers of macrophages infiltrating the islet cells, although significant numbers of T cells, and later B cells, also appear to be recruited to the site [Hanenberg et al., Diabetologia, 32:126-134 (1989 )].

Development of diabetes in BB rats appears to depend on both early macrophage infiltration and subsequent T-cell recruitment. Treatment of BB rats with silica particles, which are toxic to macrophages, has been effective in blocking the early macrophage infiltration of the islet cells. In the absence of early macrophage infiltration, subsequent tissue damage by an autoaggressive lymphocyte population does not occur. Administration of monoclonal antibody OX-19 (specific for rat CD5) or monoclonal antibody OX-8 (specific for rat CD8), which block the T-cell-associated phase of the disease, is also effective in suppressing the development of diabetes.

The central role of macrophages in the pathology of this model makes it attractive for testing inhibitors of aa function. Rats that are genetically predisposed to develop insulin-dependent diabetes are treated with monoclonal antibodies against aa or low molecular weight inhibitors, and evaluated for development of the disease. Prevention or delay of clinical onset is evidence that a«j plays a central role in macrophage damage to the islet cells.

D. Inflammatory bowel disease (Crohn's disease, ulcerative colitis)

Animal models used in the investigation of inflammatory bowel disease (IBD) are generally triggered by intrarectal administration of noxious irritants {eg. acetic acid or trinitrobenzenesulfonic acid/ethanol). Intestinal inflammation induced by these agents is the result of chemical or metabolic damage and lacks the chronic and spontaneously relapsing inflammation associated with human IBD. However, a recently described model using subserosal injections of purified peptide-glycan-polysaccharide (PG-PS) polymers from either group A or D streptococci appears to be a more physiologically relevant model for human IBD [Yamada et al., Gastroenterology , 104:759-771 (1993)].

In this model, PG-PS is injected into the subserous layer of the distal colon. The resulting inflammatory response is biphasic with an initial acute episode 3 days after injection, which is followed by a spontaneous chronic phase 3-4 weeks later. The late phase response is granulomatous in nature and results in chronic thickening, adhesions, colonic nodules and mucosal lesions. In addition to mucosal damage, PG-PS colitis often leads to arthritic anemia and granulomatous hepatitis. The effects of the disease outside the intestine make the model attractive for the investigation of Crohn's colitis in that a significant number of patients with active Crohn's disease suffer from arthritic joint disease and hepatobiliary inflammation.

Granulomatous lesions are the result of chronic inflammation that leads to the recruitment and subsequent activation of cells of the monocyte/macrophage lineage. The presence of granulomatous lesions in Crohn's disease and the above-mentioned animal model make this an attractive clinical target for ctd monoclonal antibodies or other inhibitors of eid function. Inhibitors of dd function are expected to block the formation of lesions associated with IBD, or even reverse the tissue damage observed in the disease.

E. Arthritis

Arthritis appears to be a multifactorial disease process involving many different inflammatory cell types, including neutrophils, T lymphocytes and phagocytic macrophages. Although many different arthritis models exist, preparations of streptococcal cell wall proteoglycan produce a disease most similar to the human disease.

In rats, streptococcal cell walls induce inflammation of peripheral joints, characterized by repeated episodes of disease progression followed by remission, ultimately resulting in joint destruction over a period of several months [Cromartie et al., J. Exp. Med., 146:1585- 1602 (1977); Schwab et al., Infection and Immunity, 55:4436-4442 (1991)]. During the chronic phase of the disease, mononuclear phagocytes or macrophages are believed to play a major role in the destruction of the synovium. Furthermore, agents that suppress the recruitment of macrophages into the synovium effectively reduce the inflammation and pathology characteristic of arthritis.

A central role for macrophages in synovium destruction leading to arthritis predicts that monoclonal antibodies against dd or inhibitors of ctd function may have therapeutic potential in the treatment of this disease. As in other previously described models, ctd monoclonal antibodies or low molecular weight inhibitors administered prophylactically are expected to block or moderate joint inflammation and prevent synovial destruction. Agents that interfere with ctd function may also moderate ongoing inflammation by preventing the recruitment of additional macrophages to the joint or by blocking macrophage activation. The net result will be to reverse ongoing destruction of the joint and facilitate tissue repair.

tion.

F. Multiple sclerosis

Although the pathogenesis of multiple sclerosis (MS) remains unclear, it is generally accepted that the disease is mediated by CD4<+->T cells that recognize autoantigens in the central nervous system and initiate an inflammatory cascade. The resulting immune response leads to the recruitment of additional inflammatory cells, including activated macrophages, which contribute to the disease. Experimental autoimmune encephalomyelitis (EAE) is an animal model that reproduces some aspects of MS. Recently, monoclonal antibodies that react with CD11b/CD18 [Huitinga et al., Eur. J. Immunol., 23:709-715 (1993)] present on inflammatory macrophages, shown to block both clinical and histological disease. The results suggest that monoclonal antibodies or low-molecular-weight inhibitors of ad are likely to be effective in blocking the inflammatory response in EAE. Such agents also have important therapeutic applications in the treatment of MS.

G. Immune complex alveolitis

Alveolar macrophages located in the alveolar ducts, airways, connective tissue and the pleural cavity in the lung, represent the lung's first line of defense against inhaled substances from the environment. In response to stimulation by substances, including bacterial-derived LPS, IFN-γ, and immune complexes, alveolar macrophages release many different potent inflammatory mediators, including highly reactive oxygen radicals and nitrogen intermediates. Although superoxide anions, hydrogen peroxide and nitric oxide (NO) have important functions in eradicating pathogens and lysing tumor targets, these agents can have deleterious effects on normal tissues.

In a rat model of immune complex alveolitis, NO- release from alveolar macrophages has been shown to mediate much of the lung damage [Mulligan et al., Proe. Nati. Acad. Pollock. (USA), 88:638-642 (1991)]. NO- has also been implicated as a mediator in other immune complex-mediated injuries, including dermal vasculitis [Mulligan et al., supra] and may potentially play a

role in diseases such as glomerulonephritis.

N0--mediated tissue damage is not limited to inflammation involving immune complexes. Microglial cells stimulated by agents such as PMA, LPS or IFN-γ, produce e.g. NO-i amounts capable of killing oligodendrocytes [Merrill et al., Immunol., 151:2132 (1993)]. Pancreatic islet cells have also been found to be sensitive to NO, and macrophage release of this mediator has been implicated in the tissue damage that leads to diabetes [Kroncke et al., BBRC, 175:752-758

(1991)]. Recently, it was decisively demonstrated that NO- release plays a role in endotoxic shock [MacMicking et al., Cell, 81:641-650 (1995)]. When administered lipopolysaccharide (LPS), normal wild-type mice experience a severe, progressive decrease in arterial pressure leading to death. However, mice lacking inducible nitric oxide experience a much less severe reduction in arterial pressure in response to LPS, and all survive the treatment.

In vitro assays indicate that blocking ctd is effective in blocking some aspects of macrophage activation (or leukocyte activation expressing eid in general), including NO- release. Alveolar macrophages stimulated with IFN-γ in the presence of anti-Od polyclonal antiserum (raised in rabbits against a rat Od-I domain polypeptide) were found to produce significantly less nitrite/nitrate breakdown products of NO- than macrophages treated with control antiserum. This finding indicates that monoclonal antibodies against ctd, particularly against the I domain, may be potent anti-inflammatory agents with potential applications in MS, diabetes, lung inflammation and endotoxic shock. Furthermore, unlike CD18, which effects the function of many different leukocyte types, the restricted distribution of ctd makes it a more attractive target than CD18 to prevent macrophage activation (or leukocyte activation expressing ad in general).

Rat IgG immune complex-induced alveolitis is a widely used experimental model important for understanding acute lung injury. The injury is triggered by instilling anti-bovine serum albumin (BSA) antibodies into the lungs via a cannula in the trachea, followed by an intravenous injection of BSA. The formation of immune complexes in the micro blood vessels in the lung leads to complement activation and recruitment of neutrophils to the lung. Presumably, the formation of immune complexes in the lung follows the egress of leukocytes from the blood and subsequent leukocyte movement through the lung epithelium. The subsequent release of mediators includes radicals, TNF-a and nitric oxide (N0-) from activated endothelial cells, neutrophils and macrophages that participate in the development of the disease. Pathological features of the disease include increased vascular permeability leading to edema and the presence of large numbers of erythrocytes and PMN present in the alveolar spaces.

Polyclonal antiserum specific for the I domain of cia was tested in a rat model of immune complex-induced alveolitis. The anti-ad polyclonal serum was administered via tracheal cannulation at the same time as anti-BSA was introduced into the lungs. Lung injury was then induced by intravenous administration of BSA together with a trace amount of <125>I-labeled BSA (ca. 800,000 cpm) to quantify edema as a result of the lung injury. The lung injury was allowed to develop for 4 hours, and the injury was assessed using a lung permeability value, defined as the ratio of 12<5>I-labeled BSA in the lung compared to the amount of marker present in 1.0 ml of blood. Lung permeability values for positive controls typically range between 0.6 and 0.8, while negative controls (rats not receiving BSA) have permeability index values in the range of 0.1-0.2.

Initial investigations indicated that treatment with anti-Od polyclonal antiserum reduced lung permeability values by more than 50%, representing a dramatic moderation of lung injury. Historically, treatments with anti-CD18 have reduced permeability values by 60%. These findings indicate that ctd may be the most important p2-integrin during acute lung injury, but it cannot be determined precisely whether the effect of antisera prevents leukocyte egress from the blood or movement through the lung epithelium.

As further evidence that aa moderates lung injury, TNF-a levels in the bronchoalveolar lavage fluid were evaluated. Treatment with ant i-ctd-ant in serum was found to reduce TNF-a levels approx. 4 times. TNF-α has long been considered an important mediator in acute lung inflammation and responsible for the recruitment of inflammatory cells to sites of inflammation, cell activation and tissue damage. Presumably, anti-aa antiserum blocks activation of alveolar macrophages present during the formation of immune complex alveolitis, thereby moderating the release of TNF-α and NO-, and reducing subsequent tissue damage caused by these agents and the recruitment of neutrophils.

Example 26

Expression of aa in preclinical models

To assess differential expression of aa in different disease states, tissue sections from animal disease models were stained with anti-aa polyclonal serum produced as described above (see Example 18). Tissues from normal and diseased rats were sectioned at 6 µm thicknesses and air-dried on Superfrost Plus slides (VWR Scientific) at room temperature overnight. After drying, the sections were stored at -70 °C until they were used. Before use, the slides were removed from -70 °C and placed at 50 °C for approximately 5 minutes. Sections were fixed in cold (4°C) acetone (Stephens Scientific) for 10 min at room temperature and allowed to dry at room temperature. Each section was blocked with 150 µl of a solution containing 30% normal rat serum (Harlan Bioproducts), 5% normal goat serum (Vector Laboratories), and 1% bovine serum (BSA) (Sigma Chemical Company) in 1 x TBS for 30 min at room temperature, after which the solution was carefully dried away from the sections. Rabbit polyclonal serum, at a protein concentration of 34 pg/ml, and preimmune serum from the same rabbit, at a protein concentration of 38.5 µg/ml, were diluted in the blocking solution, and 100 µl was separately applied to each tissue section for 30 minutes at 37° C. The serum solution was wiped away from the sections, and unbound antibody was removed by washing three times in 1 x TBS for 5 minutes. Excess TBS was removed by drying off after the last wash. Biotinylated goat anti-rabbit antibody from an Elite rabbit IgG-Vectastain ABC kit (Vector) was prepared according to the manufacturer's suggested protocol, and 100 µl of the resulting solution was applied to each section for 15 min at 37 °C. Slides were washed twice in 1x TBS for 5 min, after which 100 µl streptavidin-gold conjugate (Goldmark Biologicals), diluted 1:100 in 5% normal rat serum and 1% BSA, was applied to each section for 1 h at room temperature. The slides were washed three times with TBS for 5 minutes, and 100 µl of 1% glutaraldehyde (Sigma) in TBS buffer was applied for 5 minutes at room temperature. The slides were again washed three times in TBS for 5 minutes and five times in sterile, deionized water for 3 minutes. Excess fluid was wiped off each slide, and two drops of silver enhancement and initiation solution (Goldmark Biologicals) were applied to each section. The reaction was allowed to proceed for 20-30 minutes at room temperature, after which the sections were thoroughly rinsed in sterile, deionized water, air-dried overnight at room temperature and mounted with Cytoseal 60 (VWR). As controls, tissue sections were labeled with monoclonal antibodies against CDlla, CDllb, CDllc and CD18 in the same experiments using identical protocols.

Labeling with α<i polyclonal sera and monoclonal antibodies against CDlla, CDllb, CDllc and CD18 revealed a staining pattern for da that was different from that observed for the other α subunits.

In normal lung tissue, aa expression was detected on respiratory epithelium in the bronchi (but not the epithelium in the alveolar cavities) and on individual cells that appear to be alveolar macrophages in the air spaces. The signal observed with the polyclonal serum was significantly higher than the background signal with the preimmune serum control. In lung granuloma tissue, 24 and 96 hours after glycan administration, a distinct signal was detected with the Ad-staining respiratory epithelium throughout the alveolar area, and a stronger signal was detected on what appeared to be alveolar macrophages throughout the airways. In lung tissue from animals that had presumably recovered from the disease (killed 16 days after glycan administration), no signal was observed with the aa antibody. Very little background was observed with the preimmunization serum in each of

these tissues.

When using rat lung tissue from an antigen-induced asthma model, a very strong signal with cid antibody was detected in the respiratory epithelium in both the bronchi and alveolar spaces. The signal was significantly higher than the background signal in the preimmunization serum control.

Many modifications and variations of the present invention, as described in the illustrative examples above, are expected to be apparent to those skilled in the art. The present invention should thus only be limited in accordance with the accompanying claims.

Claims (2)

1. Hybridoma, characterized in that it has the designation 199M with accession number ATCC HB-12058. 2. Monoclonal antibody, characterized in that it is secreted by the hybridoma according to claim 1.